Methods of lowering serum cholesterol

ABSTRACT

Methods of treating subjects having diseases, disorders, or conditions, including disorders associated with cholesterol homeostasis, responsive to agents modulating Kupffer cell function, including methods of administration and dosing regimens associated therewith, are provided. Methods of treating subjects having liver diseases, disorders, or conditions, including non-alcoholic steatohepatitis and non-alcoholic fatty liver disease, with an IL-10 agent are also provided.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority benefit of U.S. provisional applicationSer. No. 62/006,651, filed Jun. 2, 2014, which application isincorporated herein in its entirety.

FIELD OF THE INVENTION

This invention relates to methods of treating or preventinghypercholesterolemia, and a diverse array of related diseases, disordersand conditions, by administering agents that modulate lipoproteinhomeostasis.

INTRODUCTION

Hypercholesterolemia, the presence of high levels of cholesterol in theblood, is a common form of hyperlipidemia and hyperlipoproteinemia.Cholesterol is transported in the blood plasma within lipoproteins,which are classified by their density: VLDL (very low densitylipoprotein), IDL (intermediate density lipoprotein), LDL (low densitylipoprotein), and HDL (high density lipoprotein). Elevated levels ofVLDL, IDL and LDL—LDL in particular—are associated with an increasedrisk of cardiovascular disorders, including atherosclerosis and heartdisease. Conversely, higher levels of HDL are thought to exert aprotective effect. In subjects with hypercholesterolemia uncontrolled bydietary restrictions, pharmacological intervention is frequentlywarranted.

Increased activity of the innate immune system has been linked to thepathogenesis of the dyslipidemia and insulin resistance associated withobesity and type II diabetes. Macrophages, myeloid-derived mononuclearcells, play a key role in the innate immune system. They are recruitedto tissues in response to infection, tissue damage, or other trauma, andare particularly enriched in tissues that are frequently exposed toexogenous and endogenous toxins, such as the liver. Recent studiesindicate that macrophages are involved in diet-induced alterations inhepatic liver metabolism and insulin sensitivity and suggest that theyplay a role in type II diabetes and obesity (Huang et al., Diabetes59:347-57 (2010). Thus, modulation of hepatic macrophage homeostasis mayprovide another approach for the treatment and prevention of metabolicabnormalities.

Although conventional lipid-lowering agents, which generally exert theiractivity by reducing cholesterol production or absorption, are effectivein treating most patient populations, alternative agents, especiallyagents acting through different mechanism(s) of action, would provide avaluable therapeutic option, both as monotherapy and as an addition toan existing pharmacological regimen.

SUMMARY

The present disclosure contemplates methods of using IL-10, modified(e.g., pegylated) IL-10, and associated agents described herein, andcompositions thereof, to treat and/or prevent various diseases,disorders and conditions, and/or the symptoms thereof. Particularembodiments are directed to the treatment and/or prevention ofabnormally high levels of cholesterol and/or manifestation(s) ofhypercholesterolemia in as subject. Further particular embodiments aredirected to the modulation of Kupffer cells (e.g., through increasedactivity and/or increased numbers) to effect the treatment and/orprevention of abnormally high levels of cholesterol and/ormanifestation(s) of hypercholesterolemia in as subject.

Hypercholesterolemia itself is generally asymptomatic. However, chronicelevation of serum cholesterol contributes to formation of atheromatousplaques in the arteries. Relatively small plaques may rupture and causea clot to form and obstruct blood flow. By comparison, larger plaquescan result in arterial stenosis or occlusion of the involved arteries. Asudden occlusion of a coronary artery results in a myocardialinfarction, whereas an occlusion of an artery supplying the brain canresult in a stroke.

Gradual development of the stenosis or occlusion that causes aprogressive reduction in the blood supply to the tissues and organsfrequently results in impairment of the activity thereof. Tissueischemia may manifest as one or more symptoms. For example, temporaryischemia of the brain (a transient ischemic attack) may manifest astemporary loss of vision, dizziness, or impairment of balance, aphasia,paresis and paresthesia. Insufficient blood supply to the heart maymanifest as chest pain; ischemia of the eye may manifest as transientvisual loss in one eye; and insufficient blood supply to the legs maymanifest as calf pain.

Hypercholesterolemia may be categorized into various types withcharacteristic manifestations. For example, Type IIahyperlipoproteinemia may be associated with xanthelasma palebarum(yellowish patches underneath the skin around the eyelids), arcussenilis (white or gray discoloration of the peripheral cornea), andxanthomata (deposition of yellowish cholesterol-rich material) of thetendons (usually the fingers). In contrast, Type III hyperlipidemia maybe associated with xanthomata of the palms, knees and elbows.

According to the lipid hypothesis, abnormal cholesterol levels(generally higher concentrations of LDL particles and lowerconcentrations of functional HDL particles) in the blood are stronglyassociated with cardiovascular disease due to promotion of atheromadevelopment in arteries (atherosclerosis). As high circulating LDLconcentrations have been linked to atheroma formation, LDL is oftenreferred to as “bad cholesterol”; in contrast, high concentrations ofHDL can remove cholesterol from cells, diminishing atheroma formation,and thus HDL is often referred to as “good cholesterol”. However, recentevidence suggests that total cholesterol is the most relevant indicatorof cardiovascular abnormalities.

Heretofore, the therapeutic control of systemic cholesterol levels hasfocused primarily on inhibition of the uptake of dietary cholesterol andon inhibition of endogenous hepatocellular cholesterol synthesis. By wayof example, ezetimibe (ZETIA) inhibits dietary uptake in the smallintestine, and it has been shown to dramatically decrease serumcholesterol in both genetically deficient mice strains APOE−/− andLDLR−/− mice fed a high fat diet (Davis, H. R., Jr., et al.,Arterioscler Thromb Vasc Biol, 2001. 21(12):2032-38). By way of furtherexample, the statin class of cholesterol-lowering therapeutic agents actthrough the Mevalonate pathway, which is primarily active inhepatocytes, by inhibition of HMG-CoA-mediated cholesterol synthesis.

Therapeutic modalities for the treatment of hypercholesterolemia thatact through other mechanisms of action have been developed or are inlate-stage development. These modalities include inhibitors of PCSK9,which enhance the recycling of the LDL receptor to the cell surface inorder to increase the rate at which LDL particles are removed from theblood; mipomersen (KYNAMERO), an antisense oligonucleotide used in thetreatment of familial hypercholesterolemia (FH) that acts by hybridizingto apoB-100 and thus limiting the amount of LDL-C than can be formed;and lomitapide (JUXTAPID), also used in the treatment of FH, whichprevents the formation and secretion of VLDL by inhibiting themicrosomal triglyceride transfer protein in the liver. As with othercholesterol-lowering agents, these modalities are associated withadverse effects that limit their utility in certain patient populations(e.g., mipomersen and lomitapide have been associated with fatty liverdisease caused by the accumulation of cholesterol in the liver).

As discussed further herein, macrophages play a large role incholesterol homeostasis, through the uptake of LDL cholesterol, as wellas the Ac-LDL and Ox-LDL forms of cholesterol. Although Kupffer Cells(KCs) only represent approximately 10-15% of the total 10-30 billionliver cells, KCs are 18 times more efficient in cholesterol catabolismthan hepatocytes. Thus, modulation of KCs function and/or an increase inKCs number represent a novel avenue for the treatment and prevention ofhypercholesterolemia and associated diseases, disorders and conditions.Embodiments of the current disclosure comprise the administration of anagent (e.g., a small molecule, a polypeptide, or an antibody) to asubject that modulates Kupffer cells (e.g., through increased activityand/or increased numbers) to effect the treatment and/or prevention ofabnormally high levels of cholesterol and/or manifestation(s) ofhypercholesterolemia in as subject. In particular embodiments,modulation of Kupffer cell function is used in the treatment and/orprevention of familial hypercholesterolemia (FH). In certainembodiments, the agent is an IL-10 agent (e.g., PEG-IL-10).

As discussed further hereafter, human IL-10 is a homodimer and eachmonomer comprises 178 amino acids, the first 18 of which comprise asignal peptide. Particular embodiments of the present disclosurecomprise mature human IL-10 polypeptides lacking the signal peptide(see, e.g., U.S. Pat. No. 6,217,857), or mature human PEG-IL-10. Infurther particular embodiments, the IL-10 agent is a variant of maturehuman IL-10. The variant may exhibit activity less than, comparable to,or greater than the activity of mature human IL-10; in certainembodiments the activity is comparable to or greater than the activityof mature human IL-10.

The terms “IL-10”, “IL-10 polypeptide(s),” “agent(s)” and the like areintended to be construed broadly and include, for example, human andnon-human IL-10-related polypeptides, including homologs, variants(including muteins), and fragments thereof, as well as IL-10polypeptides having, for example, a leader sequence (e.g., the signalpeptide), and modified versions of the foregoing. In further particularembodiments, the terms “IL-10”, “IL-10 polypeptide(s), “agent(s)” areagonists. Particular embodiments relate to pegylated IL-10, which isalso referred to herein as “PEG-IL-10”.

The present disclosure contemplates methods wherein the IL-10 agentcomprises at least one modification to form a modified IL-10 agent,wherein the modification does not alter the amino acid sequence of theIL-10 agent. Certain embodiments of the present disclosure contemplatesuch modifications in order to enhance one or more properties (e.g.,pharmacokinetic parameters, efficacy, etc.). In further embodiments,modification of IL-10 does not result in a therapeutically relevant,detrimental effect on immunogenicity, and in still further embodimentsmodified IL-10 is less immunogenic than unmodified IL-10. In someembodiments, the modified IL-10 agent is a PEG-IL-10 agent. ThePEG-IL-10 agent may comprise at least one PEG molecule covalentlyattached to at least one amino acid residue of at least one subunit ofIL-10 or comprise a mixture of mono-pegylated and di-pegylated IL-10 inother embodiments. The PEG component of the PEG-IL-10 agent may have amolecular mass greater than about 5 kDa, greater than about 10 kDa,greater than about 15 kDa, greater than about 20 kDa, greater than about30 kDa, greater than about 40 kDa, or greater than about 50 kDa. In someembodiments, the molecular mass is from about 5 kDa to about 10 kDa,from about 5 kDa to about 15 kDa, from about 5 kDa to about 20 kDa, fromabout 10 kDa to about 15 kDa, from about 10 kDa to about 20 kDa, fromabout 10 kDa to about 25 kDa or from about 10 kDa to about 30 kDa.

Additional modified IL-10 agents are discussed in detail hereafter. Insome embodiments, the modified IL-10 agent comprises at least one Fcfusion molecule, at least one serum albumin (e.g., HSA or BSA), an HSAfusion molecule or an albumin conjugate. In additional embodiments, themodified IL-10 agent is glycosylated, is hesylated, or comprises atleast one albumin binding domain. Some modified IL-10 agents maycomprise more than one type of modification. In particular embodiments,the modification is site-specific, and in still others it comprises alinker.

The present disclosure also contemplates nucleic acid molecules encodingthe foregoing. Certain embodiments envisage the use of gene therapy inconjunction with the teachings herein. For gene therapy uses andmethods, a cell in a subject can be transformed with a nucleic acid thatencodes an IL-10-related polypeptide as set forth herein in vivo.Alternatively, a cell can be transformed in vitro with a transgene orpolynucleotide, and then transplanted into a tissue of the subject inorder to effect treatment. In addition, a primary cell isolate or anestablished cell line can be transformed with a transgene orpolynucleotide that encodes an IL-10-related polypeptide, and thenoptionally transplanted into a tissue of a subject.

As delineated in the Experimental section, PEG-rMuIL-10 was found todecrease physiological plasma cholesterol levels by up to 70% inaggressively-challenged high fat diet-fed LDLR−/− mice in a phagocytoticcell-dependent manner. This finding, consistent between mice and humans,demonstrates the nexus between IL-10's regulation of KC scavengerreceptor modulation and the enhancement of cholesterol uptake. Moreover,phagocytotic cells exert a consistent and powerful role in the normalendogenous regulation of total plasma cholesterol.

In particular embodiments, the present disclosure is drawn to a methodof identifying an agent that induces phosphorylation of STAT3 in a KC,comprising: a) contacting a candidate agent with a KC, b) determiningthe level of STAT3 phosphorylation in the KC, and c) comparing the levelof STAT3 phosphorylation in b) with the level of STAT3 phosphorylationinduced by a reference standard, wherein a higher level of STAT3phosphorylation in the KC compared to the level of STAT3 phosphorylationin the reference standard identifies the candidate agent as an agentthat induces phosphorylation.

In some embodiments, an vitro model is used in identifying an agent thatinduces phosphorylation of STAT3 in a KC. In other embodiments, the KCis from a sinusoid of the liver.

In further embodiments of the present disclosure, the candidate agentcomprises a small molecule, a polypeptide or an antibody.

The present disclosure also contemplates embodiments wherein thereference standard is an interleukin, an interferon, epidermal growthfactor (EGF), hepatocyte growth factor (HGF), leukemia inhibitory factor(LIF), bone morphogenetic protein 2 (BMP-2), oncostatin M (OSM), orleptin. In particular embodiments, the interleukin is IL-5, IL-6 orIL-10.

The present disclosure further comprises methods of evaluating whetheran agent that induces phosphorylation reduces at least one of serumcholesterol levels and triglyceride levels. In some methods, suchevaluating is conducted with a biochemical assay, an in vitro assay, anex vivo assay or an in vivo model.

The present disclosure also contemplates methods of identifying an agentthat lowers serum cholesterol in a subject, comprising: a) administeringa candidate agent to the subject, wherein the candidate agent inducesSTAT3 phosphorylation in a KC, b) determining the level of serumcholesterol in the subject, and c) comparing the level of serumcholesterol in b) with the level of serum cholesterol measured afteradministering a reference standard (e.g., a statin) to the subject,wherein the reference standard is known to lower serum cholesterol;wherein a candidate agent that lowers serum cholesterol more than orcomparable to the reference standard identifies an agent that lowersserum cholesterol in the subject. In some embodiments of the presentdisclosure, the candidate agent comprises a small molecule, apolypeptide, or an antibody. Additional embodiments of the presentdisclosure further comprise evaluating whether the agent that lowersserum cholesterol in the subject induces hepatocyte proliferation.

Embodiments are contemplated wherein the subject is a human. The serumcholesterol level in a human may be from 200 to 239 mg/dL or at least240 mg/dL. Further embodiments are contemplated wherein the animal modelis a mouse model (e.g., an LDLR−/− mouse model).

The present disclosure also contemplates methods of lowering serumcholesterol in a subject in need thereof, comprising administering atherapeutically effective amount of an agent that modulates KChomeostasis. The KC homeostasis can comprise increasing the capacity ofthe KCs to remove lipoproteins from the serum, and/or it can compriseincreasing the number of KCs removing lipoproteins from the serum.

An agent referenced in the preceding paragraph may be any agentidentified using one of the aforementioned methods. In particularembodiments the agent is an IL-10 agent. These agents may beadministered to a subject parenterally (e.g., subcutaneously), orally,or by any other means described herein or known to the skilled artisan.

Combination therapy is contemplated herein such that a therapeuticallyacceptable amount of at least one additional cholesterol-lowering agentis administered.

The present disclosure contemplates methods of treating or preventinghypercholesterolemia or a hypercholesterolemia-associated disease,disorder or condition in a subject (e.g., a human), comprisingadministering (for example, via parenteral (e.g., SC) or oraladministration) to the subject a therapeutically effective amount of anagent identified using any of the methods described herein. Thehypercholesterolemia-associated disease, disorder or condition can be acardiovascular disorder (e.g., atherosclerosis), thrombosis or athrombotic condition, an inflammatory disorder (e.g., vasculitis), or afibrotic disorder. Specific embodiments are contemplated wherein afibrotic disorder is hepatic-related, such as non-alcoholic fatty liverdisease (NAFLD), non-alcoholic steatohepatitis (NASH) or cirrhosis.

In particular embodiments, methods can further comprise administering atherapeutically acceptable amount of at least one additional therapeuticor prophylactic agent, such as a cholesterol homeostasis agent (e.g., astatin, a bile acid resin, ezetimibe, a fibric acid, a niacin, or aPCSK9 inhibitor), an anti-obesity agent, or an anti-inflammatory agent.Other agents are contemplated herein for use in combination therapy, andthese agents are known to those of ordinary skill in the art.

The present disclosure contemplates pharmaceutical compositionscomprising a pharmaceutically effective amount of one or more of theaforementioned agents and a pharmaceutically acceptable diluent, carrieror excipient. Generally, such compositions are suitable for humanadministration. These pharmaceutical compositions may comprise one ormore additional prophylactic or therapeutic agents, examples of whichare described herein.

In certain embodiments, a sterile container (e.g., a vial or a syringe)may contain these pharmaceutical compositions. The sterile containersmay be housed in a kit, which may also contain one or more additionalprophylactic or therapeutic agent(s), means for reconstitution,directions for use, etc.

The present disclosure also contemplates methods of treating orpreventing a liver disease, disorder or condition in a subject (e.g., ahuman), comprising administering (e.g., parenterally, includingsubcutaneously) to the subject a therapeutically effective amount of anIL-10 agent that modulates KC homeostasis, wherein the liver diseasedisorder or condition is non-alcoholic steatohepatitis (NASH) ornon-alcoholic fatty liver disease (NAFLD).

Further embodiments of the present disclosure contemplate methods oftreating or preventing a liver disease, disorder or condition in asubject (e.g., a human), comprising administering (e.g., parenterally,including subcutaneously) to the subject a therapeutically effectiveamount of an IL-10 agent that modulates KC homeostasis, wherein theamount is sufficient to achieve a mean IL-10 serum trough concentrationfrom 1 pg/mL to 10.0 ng/mL; and wherein the liver disease disorder orcondition is NASH or NAFLD.

In still further embodiments, the present disclosure contemplatesmethods of treating or preventing a liver disease, disorder or conditionin a subject (e.g., a human), comprising administering (e.g.,parenterally, including subcutaneously) to the subject a therapeuticallyeffective amount of a cytokine (e.g., an IL-10 agent) that modulates KChomeostasis, wherein the amount is sufficient to maintain a meancytokine (e.g., IL-10) serum trough concentration over a period of time;wherein the mean cytokine (e.g., IL-10) serum trough concentration isfrom 1.0 pg/mL to 10.0 ng/mL; wherein the mean cytokine (e.g., IL-10)serum trough concentration is maintained for at least 95% of the periodof time; and wherein the liver disease disorder or condition is NASH orNAFLD. As used herein, the term “cytokine(s)” is meant to have itsordinary meaning in the art.

In certain methods, the mean cytokine (e.g., IL-10) serum troughconcentration is in the range of from 0.1 ng/mL to 10 ng/mL, from 0.1ng/mL to 5.5 ng/mL, from 0.5 ng/mL to 10 ng/mL, from 0.5 ng/mL to 5.5ng/mL, from 0.75 ng/mL to 10.0 ng/mL, from 0.75 ng/mL to 5.5 ng/mL, from0.9 ng/mL to 10.0 ng/mL, from 0.9 ng/mL to 5.5 ng/mL, from 0.9 ng/mL to5.1 ng/mL, from 0.9 ng/mL to 5.0 ng/mL, from 0.9 ng/mL to 4.5 ng/mL,from 0.9 ng/mL to 4.0 ng/mL, from 0.9 ng/mL to 3.5 ng/mL, from 0.9 ng/mLto 3.0 ng/mL, from 1.0 ng/mL to 5.1 ng/mL, from 1.0 ng/mL to 5.0 ng/mL,from 1.0 ng/mL to 4.5 ng/mL, from 1.0 ng/mL to 4.0 ng/mL, from 1.0 ng/mLto 3.5 ng/mL, or from 1.0 ng/mL to 3.0 ng/mL. The present disclosurecontemplates methods wherein the cytokine (e.g., an IL-10 agent) isadministered to the subject at least twice daily, at least once daily,at least once every 48 hours, at least once every 72 hours, at leastonce weekly, at least once every 2 weeks, at least once monthly, atleast once every 2 months, or at least once every 3 months, or lessfrequent. In some embodiments of the methods described herein, the meanIL-10 serum trough concentration is maintained for at least 90% of theperiod of time, for at least 95% of the period of time, for at least 97%of the period of time, for at least 99% of the period of time, or for100% of the period of time.

The present disclosure contemplates embodiments wherein the IL-10 agentis mature human IL-10 or a variant of mature human IL-10. In particularembodiments, the variant exhibits activity comparable to the activity ofmature human IL-10.

In some embodiments, the disease, disorder or condition is NASH, and inother embodiments it is NAFLD.

The modulation of Kupffer cell homeostasis comprises increasing thecapacity of KCs to remove lipoproteins from the serum in someembodiments, and increasing the number of KCs to remove lipoproteinsfrom the serum in others.

Embodiments are contemplated herein wherein the cytokine (e.g., an IL-10agent) decreases hepatic cholesterol and/or triglycerides, decreases orreverses peri-portal collagen deposition, or increases the number ofhepatocytes.

Some embodiments also comprise administering the cytokine (e.g., anIL-10 agent) with at least one additional prophylactic or therapeuticagent. In certain embodiments of the present disclosure, theprophylactic or therapeutic agent is a cholesterol homeostasis agent. Insome embodiments, the cholesterol homeostasis agent comprises a statin,a bile acid resin, ezetimibe, a fibric acid, a niacin, or a PCSK9inhibitor. The cholesterol hemostasis agent frequently improves, eitherdirectly or indirectly, a cardiovascular disorder. In particularembodiments, a prophylactic or therapeutic agent is one useful in theprevention or treatment of atherosclerosis. In additional embodiments,the prophylactic or therapeutic agent is an anti-diabetic agent or ananti-obesity agent, whereas in other embodiments it is an immune agentor an anti-inflammatory agent. Additional exemplary prophylactic andtherapeutic agents are set forth hereafter.

Other embodiments of the present disclosure are described herein, whilestill others would be envisaged by the skilled artisan after reviewingthis disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1L illustrate the regulatory effect of PEG-rMuIL-10 on plasmacholesterol levels in wild-type and LDLR−/− mice fed a normal and highfat diet.

FIGS. 1M-1N illustrate the effect of PEG-rHuIL-10 on plasma cholesterollevels in oncology patients.

FIGS. 2A-2L illustrate the effect of PEG-rMuIL-10 on the expression ofgenes associated with liver function and cholesterol regulation.

FIGS. 3A-3H illustrate the effect of PEG-rMuIL-10 on scavenger receptorsand the number of Kupffer cells in treated and untreated liver tissue.

FIGS. 4A-4G illustrate the results of an assessment conducted in orderto determine whether cells associated with the myeloid lineage wereresponsible for the control of plasma cholesterol by PEG-rMuIL-10.

FIGS. 5A-5D illustrate the results of an assessment conducted in orderto determine which cells in the liver respond to PEG-rHuIL-10.

FIGS. 6A-6F illustrate that PEG-rHuIL-10 increases the uptake ofacetylated LDL (Ac-LDL) and oxidized LDL (Ox-LDL) in monocytes, and theupdate of LDL in Kupffer cells.

FIGS. 7A-7C illustrate that the effect on cholesterol lowering ofPEG-rMuIL-10 and Ezetimibe is additive.

FIGS. 8A-8I illustrate that Kupffer cells play a role in the reductionof the accumulation of lipids, cholesterol and triglycerides observedwith the introduction of PEG-rMuIL-10.

FIGS. 9A-9I illustrate that treatment of animals having the indicatedbackgrounds with PEG-rMuIL-10 resulted in hepatocyte proliferation.

DETAILED DESCRIPTION

Before the present disclosure is further described, it is to beunderstood that the disclosure is not limited to the particularembodiments set forth herein, and it is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meaning as commonly understood by one of ordinary skill in theart to which this invention belongs.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology such as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Further,the dates of publication provided may be different from the actualpublication dates, which may need to be independently confirmed.

Overview

The present disclosure contemplates the use of the agents describedherein, and compositions thereof, to treat and/or prevent variousmetabolic-related diseases (e.g., hypercholesterolemia), disorders andconditions, and/or the symptoms thereof.

The present disclosure also contemplates the use of IL-10 agents (e.g.,an IL-10 polypeptide) and other cytokines to treat or prevent a liverdisease, disorder or condition comprising administering an IL-10 agent(or other cytokine agents) that modulates Kupffer cell homeostasis. Inparticular embodiments, the liver disease disorder or condition isnon-alcoholic steatohepatitis (NASH) or non-alcoholic fatty liverdisease (NAFLD).

In certain aspects of the present disclosure, such treatment orprevention is effected by utilizing particular dosing parameters. Thepresent disclosure is based on the findings that there is an optimalmean IL-10 (or other cytokine) serum trough concentration range and anoptimal dosing range that achieves therapeutically relevant reduction ofserum cholesterol.

In some embodiments of the present disclosure, a subject having, or atrisk of having, a disease or disorder treatable by an IL-10 agent (orother cytokine agent) is administered the IL-10 agent in an amountsufficient to achieve a serum trough concentration greater than about 1ng/mL but less than about 10 ng/mL, whereas in other embodiments theserum trough concentration is greater than about 2 ng/mL but less thanabout 10 ng/mL.

Some of the embodiments and descriptions set forth herein are describedin the context of an IL-10 agent (e.g., a PEG-IL-10 agent). It is to beunderstood that, when appropriate in view of the context in which it isbeing used, recitation of an IL-10 agent may also refer more broadly toa cytokine agent.

It should be noted that any reference to “human” in connection with thepolypeptides and nucleic acid molecules of the present disclosure is notmeant to be limiting with respect to the manner in which the polypeptideor nucleic acid is obtained or the source, but rather is only withreference to the sequence as it may correspond to a sequence of anaturally occurring human polypeptide or nucleic acid molecule. Inaddition to the human polypeptides and the nucleic acid molecules whichencode them, the present disclosure contemplates IL-10-relatedpolypeptides and corresponding nucleic acid molecules (and, in certaininstances, cytokine polypeptides and corresponding nucleic acidmolecules) from other species.

Definitions

Unless otherwise indicated, the following terms are intended to have themeaning set forth below. Other terms are defined elsewhere throughoutthe specification.

The terms “patient” or “subject” are used interchangeably to refer to ahuman or a non-human animal (e.g., a mammal).

The terms “administration”, “administer” and the like, as they apply to,for example, a subject, cell, tissue, organ, or biological fluid, referto contact of, for example, IL-10 or PEG-IL-10), a nucleic acid (e.g., anucleic acid encoding native human IL-10); a pharmaceutical compositioncomprising the foregoing, or a diagnostic agent to the subject, cell,tissue, organ, or biological fluid. In the context of a cell,administration includes contact (e.g., in vitro or ex vivo) of a reagentto the cell, as well as contact of a reagent to a fluid, where the fluidis in contact with the cell.

The terms “treat”, “treating”, treatment” and the like refer to a courseof action (such as administering IL-10 or a pharmaceutical compositioncomprising IL-10) initiated after a disease, disorder or condition, or asymptom thereof, has been diagnosed, observed, and the like so as toeliminate, reduce, suppress, mitigate, or ameliorate, either temporarilyor permanently, at least one of the underlying causes of a disease,disorder, or condition afflicting a subject, or at least one of thesymptoms associated with a disease, disorder, condition afflicting asubject. Thus, treatment includes inhibiting (e.g., arresting thedevelopment or further development of the disease, disorder or conditionor clinical symptoms association therewith) an active disease. The termsmay also be used in other contexts, such as situations where IL-10 orPEG-IL-10 contacts an IL-10 receptor in, for example, the fluid phase orcolloidal phase.

The term “in need of treatment” as used herein refers to a judgment madeby a physician or other caregiver that a subject requires or willbenefit from treatment. This judgment is made based on a variety offactors that are in the realm of the physician's or caregiver'sexpertise.

The terms “prevent”, “preventing”, “prevention” and the like refer to acourse of action (such as administering IL-10 or a pharmaceuticalcomposition comprising IL-10) initiated in a manner (e.g., prior to theonset of a disease, disorder, condition or symptom thereof) so as toprevent, suppress, inhibit or reduce, either temporarily or permanently,a subject's risk of developing a disease, disorder, condition or thelike (as determined by, for example, the absence of clinical symptoms)or delaying the onset thereof, generally in the context of a subjectpredisposed to having a particular disease, disorder or condition. Incertain instances, the terms also refer to slowing the progression ofthe disease, disorder or condition or inhibiting progression thereof toa harmful or otherwise undesired state.

The term “in need of prevention” as used herein refers to a judgmentmade by a physician or other caregiver that a subject requires or willbenefit from preventative care. This judgment is made based on a varietyof factors that are in the realm of a physician's or caregiver'sexpertise.

The phrase “therapeutically effective amount” refers to theadministration of an agent to a subject, either alone or as part of apharmaceutical composition and either in a single dose or as part of aseries of doses, in an amount capable of having any detectable, positiveeffect on any symptom, aspect, or characteristic of a disease, disorderor condition when administered to the subject. The therapeuticallyeffective amount can be ascertained by measuring relevant physiologicaleffects, and it can be adjusted in connection with the dosing regimenand diagnostic analysis of the subject's condition, and the like. By wayof example, measurement of the amount of inflammatory cytokines producedfollowing administration may be indicative of whether a therapeuticallyeffective amount has been used.

The phrase “in a sufficient amount to effect a change” means that thereis a detectable difference between a level of an indicator measuredbefore (e.g., a baseline level) and after administration of a particulartherapy. Indicators include any objective parameter (e.g., serumconcentration of IL-10) or subjective parameter (e.g., a subject'sfeeling of well-being).

The term “small molecules” refers to chemical compounds having amolecular weight that is less than about 10 kDa, less than about 2 kDa,or less than about 1 kDa. Small molecules include, but are not limitedto, inorganic molecules, organic molecules, organic molecules containingan inorganic component, molecules comprising a radioactive atom, andsynthetic molecules. Therapeutically, a small molecule may be morepermeable to cells, less susceptible to degradation, and less likely toelicit an immune response than large molecules.

The term “ligand” refers to, for example, a peptide, a polypeptide, amembrane-associated or membrane-bound molecule, or a complex thereof,that can act as an agonist or antagonist of a receptor. “Ligand”encompasses natural and synthetic ligands, e.g., cytokines, cytokinevariants, analogs, muteins, and binding compositions derived fromantibodies. “Ligand” also encompasses small molecules, e.g., peptidemimetics of cytokines and peptide mimetics of antibodies. The term alsoencompasses an agent that is neither an agonist nor antagonist, but thatcan bind to a receptor without significantly influencing its biologicalproperties (e.g., signaling or adhesion). Moreover, the term includes amembrane-bound ligand that has been changed, e.g., by chemical orrecombinant methods, to a soluble version of the membrane-bound ligand.A ligand or receptor may be entirely intracellular, that is, it mayreside in the cytosol, nucleus, or some other intracellular compartment.The complex of a ligand and receptor is termed a “ligand-receptorcomplex”.

The terms “inhibitors” and “antagonists”, or “activators” and “agonists”refer to inhibitory or activating molecules, respectively, for example,for the activation of, e.g., a ligand, receptor, cofactor, gene, cell,tissue, or organ. Inhibitors are molecules that decrease, block,prevent, delay activation, inactivate, desensitize, or down-regulate,e.g., a gene, protein, ligand, receptor, or cell. Activators aremolecules that increase, activate, facilitate, enhance activation,sensitize, or up-regulate, e.g., a gene, protein, ligand, receptor, orcell. An inhibitor may also be defined as a molecule that reduces,blocks, or inactivates a constitutive activity. An “agonist” is amolecule that interacts with a target to cause or promote an increase inthe activation of the target. An “antagonist” is a molecule that opposesthe action(s) of an agonist. An antagonist prevents, reduces, inhibits,or neutralizes the activity of an agonist, and an antagonist can alsoprevent, inhibit, or reduce constitutive activity of a target, e.g., atarget receptor, even where there is no identified agonist.

The terms “modulate”, “modulation” and the like refer to the ability ofa molecule (e.g., an activator or an inhibitor) to increase or decreasethe function or activity of an agent (e.g., an IL-10 agent) (or thenucleic acid molecules encoding them), either directly or indirectly; orto enhance the ability of a molecule to produce an effect comparable tothat of an agent (e.g., an IL-10 agent). The term “modulator” is meantto refer broadly to molecules that can effect the activities describedabove. By way of example, a modulator of, e.g., a gene, a receptor, aligand, or a cell, is a molecule that alters an activity of the gene,receptor, ligand, or cell, where activity can be activated, inhibited,or altered in its regulatory properties. A modulator may act alone, orit may use a cofactor, e.g., a protein, metal ion, or small molecule.The term “modulator” includes agents that operate through the samemechanism of action as an agent (e.g., an IL-10 agent) (i.e., agentsthat modulate the same signaling pathway as an agent (e.g., an IL-10agent) in a manner analogous thereto) and are capable of eliciting abiological response comparable to (or greater than) that of an agent(e.g., an IL-10 agent).

Examples of modulators include small molecule compounds and otherbioorganic molecules. Numerous libraries of small molecule compounds(e.g., combinatorial libraries) are commercially available and can serveas a starting point for identifying a modulator. The skilled artisan isable to develop one or more assays (e.g., biochemical or cell-basedassays) in which such compound libraries can be screened in order toidentify one or more compounds having the desired properties;thereafter, the skilled medicinal chemist is able to optimize such oneor more compounds by, for example, synthesizing and evaluating analogsand derivatives thereof. Synthetic and/or molecular modeling studies canalso be utilized in the identification of an Activator.

The “activity” of a molecule may describe or refer to the binding of themolecule to a ligand or to a receptor; to catalytic activity; to theability to stimulate gene expression or cell signaling, differentiation,or maturation; to antigenic activity; to the modulation of activities ofother molecules; and the like. The term may also refer to activity inmodulating or maintaining cell-to-cell interactions (e.g., adhesion), oractivity in maintaining a structure of a cell (e.g., a cell membrane).“Activity” can also mean specific activity, e.g., [catalyticactivity]/[mg protein], or [immunological activity]/[mg protein],concentration in a biological compartment, or the like. The term“proliferative activity” encompasses an activity that promotes, that isnecessary for, or that is specifically associated with, for example,normal cell division, as well as cancer, tumors, dysplasia, celltransformation, metastasis, and angiogenesis.

As used herein, “comparable”, “comparable activity”, “activitycomparable to”, “comparable effect”, “effect comparable to”, and thelike are relative terms that can be viewed quantitatively and/orqualitatively. The meaning of the terms is frequently dependent on thecontext in which they are used. By way of example, two agents that bothactivate a receptor can be viewed as having a comparable effect from aqualitative perspective, but the two agents can be viewed as lacking acomparable effect from a quantitative perspective if one agent is onlyable to achieve 20% of the activity of the other agent as determined inan art-accepted assay (e.g., a dose-response assay) or in anart-accepted animal model. When comparing one result to another result(e.g., one result to a reference standard), “comparable” frequentlymeans that one result deviates from a reference standard by less than35%, by less than 30%, by less than 25%, by less than 20%, by less than15%, by less than 10%, by less than 7%, by less than 5%, by less than4%, by less than 3%, by less than 2%, or by less than 1%. In particularembodiments, one result is comparable to a reference standard if itdeviates by less than 15%, by less than 10%, or by less than 5% from thereference standard. By way of example, but not limitation, the activityor effect may refer to efficacy, stability, solubility, orimmunogenicity.

The term “response,” for example, of a cell, tissue, organ, or organism,encompasses a change in biochemical or physiological behavior, e.g.,concentration, density, adhesion, or migration within a biologicalcompartment, rate of gene expression, or state of differentiation, wherethe change is correlated with activation, stimulation, or treatment, orwith internal mechanisms such as genetic programming. In certaincontexts, the terms “activation”, “stimulation”, and the like refer tocell activation as regulated by internal mechanisms, as well as byexternal or environmental factors; whereas the terms “inhibition”,“down-regulation” and the like refer to the opposite effects.

The terms “polypeptide,” “peptide,” and “protein”, used interchangeablyherein, refer to a polymeric form of amino acids of any length, whichcan include genetically coded and non-genetically coded amino acids,chemically or biochemically modified or derivatized amino acids, andpolypeptides having modified polypeptide backbones. The terms includefusion proteins, including, but not limited to, fusion proteins with aheterologous amino acid sequence; fusion proteins with heterologous andhomologous leader sequences; fusion proteins with or without N-terminusmethionine residues; fusion proteins with immunologically taggedproteins;

and the like.

It will be appreciated that throughout this disclosure reference is madeto amino acids according to the single letter or three letter codes. Forthe reader's convenience, the single and three letter amino acid codesare provided below:

G Glycine Gly P Proline Pro A Alanine Ala V Valine Val L Leucine Leu IIsoleucine Ile M Methionine Met C Cysteine Cys F Phenylalanine Phe YTyrosine Tyr W Tryptophan Trp H Histidine His K Lysine Lys R ArginineArg Q Glutamine Gln N Asparagine Asn E Glutamic Acid Glu D Aspartic AcidAsp S Serine Ser T Threonine Thr

As used herein, the term “variant” encompasses naturally-occurringvariants and non-naturally-occurring variants. Naturally-occurringvariants include homologs (polypeptides and nucleic acids that differ inamino acid or nucleotide sequence, respectively, from one species toanother), and allelic variants (polypeptides and nucleic acids thatdiffer in amino acid or nucleotide sequence, respectively, from oneindividual to another within a species). Non-naturally-occurringvariants include polypeptides and nucleic acids that comprise a changein amino acid or nucleotide sequence, respectively, where the change insequence is artificially introduced (e.g., muteins); for example, thechange is generated in the laboratory by human intervention (“hand ofman”). Thus, herein a “mutein” refers broadly to mutated recombinantproteins that usually carry single or multiple amino acid substitutionsand are frequently derived from cloned genes that have been subjected tosite-directed or random mutagenesis, or from completely synthetic genes.

The terms “DNA”, “nucleic acid”, “nucleic acid molecule”,“polynucleotide” and the like are used interchangeably herein to referto a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Non-limiting examples of polynucleotides include linear and circularnucleic acids, messenger RNA (mRNA), complementary DNA (cDNA),recombinant polynucleotides, vectors, probes, primers and the like.

As used herein in the context of the structure of a polypeptide,“N-terminus” (or “amino terminus”) and “C-terminus” (or “carboxylterminus”) refer to the extreme amino and carboxyl ends of thepolypeptide, respectively, while the terms “N-terminal” and “C-terminal”refer to relative positions in the amino acid sequence of thepolypeptide toward the N-terminus and the C-terminus, respectively, andcan include the residues at the N-terminus and C-terminus, respectively.“Immediately N-terminal” or “immediately C-terminal” refers to aposition of a first amino acid residue relative to a second amino acidresidue where the first and second amino acid residues are covalentlybound to provide a contiguous amino acid sequence.

“Derived from”, in the context of an amino acid sequence orpolynucleotide sequence (e.g., an amino acid sequence “derived from” anIL-10 polypeptide), is meant to indicate that the polypeptide or nucleicacid has a sequence that is based on that of a reference polypeptide ornucleic acid (e.g., a naturally occurring IL-10 polypeptide or anIL-10-encoding nucleic acid), and is not meant to be limiting as to thesource or method in which the protein or nucleic acid is made. By way ofexample, the term “derived from” includes homologs or variants ofreference amino acid or DNA sequences.

In the context of a polypeptide, the term “isolated” refers to apolypeptide of interest that, if naturally occurring, is in anenvironment different from that in which it may naturally occur.“Isolated” is meant to include polypeptides that are within samples thatare substantially enriched for the polypeptide of interest and/or inwhich the polypeptide of interest is partially or substantiallypurified. Where the polypeptide is not naturally occurring, “isolated”indicates that the polypeptide has been separated from an environment inwhich it was made by either synthetic or recombinant means.

“Enriched” means that a sample is non-naturally manipulated (e.g., by ascientist) so that a polypeptide of interest is present in a) a greaterconcentration (e.g., at least 3-fold greater, at least 4-fold greater,at least 8-fold greater, at least 64-fold greater, or more) than theconcentration of the polypeptide in the starting sample, such as abiological sample (e.g., a sample in which the polypeptide naturallyoccurs or in which it is present after administration), or b) aconcentration greater than that of the environment in which thepolypeptide was made (e.g., as in a bacterial cell).

“Substantially pure” indicates that a component (e.g., a polypeptide)makes up greater than about 50% of the total content of the composition,and typically greater than about 60% of the total polypeptide content.More typically, “substantially pure” refers to compositions in which atleast 75%, at least 85%, at least 90% or more of the total compositionis the component of interest. In some cases, the polypeptide will makeup greater than about 90%, or greater than about 95% of the totalcontent of the composition.

The terms “specifically binds” or “selectively binds”, when referring toa ligand/receptor, antibody/antigen, or other binding pair, indicates abinding reaction which is determinative of the presence of the proteinin a heterogeneous population of proteins and other biologics. Thus,under designated conditions, a specified ligand binds to a particularreceptor and does not bind in a significant amount to other proteinspresent in the sample. The antibody, or binding composition derived fromthe antigen-binding site of an antibody, of the contemplated methodbinds to its antigen, or a variant or mutein thereof, with an affinitythat is at least two-fold greater, at least ten times greater, at least20-times greater, or at least 100-times greater than the affinity withany other antibody, or binding composition derived therefrom. In aparticular embodiment, the antibody will have an affinity that isgreater than about 10⁹ liters/mol, as determined by, e.g., Scatchardanalysis (Munsen, et al. 1980 Analyt. Biochem. 107:220-239).

IL-10 and PEG-IL-10

The anti-inflammatory cytokine IL-10, also known as human cytokinesynthesis inhibitory factor (CSIF), is classified as a type (class)-2cytokine, a set of cytokines that includes IL-19, IL-20, IL-22, IL-24(Mda-7), and IL-26, interferons (IFN-α, -β, -γ, -δ, -ε, -κ, -Ω, and -τ)and interferon-like molecules (limitin, IL-28A, IL-28B, and IL-29).

IL-10 is a cytokine with pleiotropic effects in immunoregulation andinflammation. Although predominantly expressed in macrophages, IL-10expression has also been detected in activated T cells, B cells, mastcells, and monocytes. It is produced by mast cells, counteracting theinflammatory effect that these cells have at the site of an allergicreaction. While IL-10 predominantly limits the production and secretionof pro-inflammatory cytokines in response to toll-like receptoragonists, it is also stimulatory towards certain T cells and mast cellsand stimulates B-cell maturation, proliferation and antibody production.IL-10 can block NF-κB activity and is involved in the regulation of theJAK-STAT signaling pathway. It also induces the cytotoxic activity ofCD8+ T-cells and the antibody production of B-cells, and it suppressesmacrophage activity and tumor-promoting inflammation. The regulation ofCD8+ T-cells is dose-dependent, wherein higher doses induce strongercytotoxic responses.

As a result of its pleiotropic activity, IL-10 has been linked to abroad range of diseases, disorders and conditions, includinginflammatory conditions, immune-related disorders, fibrotic disorders,metabolic disorders, including regulation of cholesterol, and cancer.Clinical and pre-clinical evaluations with IL-10 for a number of suchdiseases, disorders and conditions have solidified its therapeuticpotential.

Human IL-10 is a homodimer with a molecular mass of 37 kDa, wherein each18.5 kDa monomer comprises 178 amino acids, the first 18 of whichcomprise a signal peptide. Each monomer comprises four cysteine residuesthat form two intramolecular disulfide bonds. The IL-10 dimer becomesbiologically inactive upon disruption of the non-covalent interactionsbetween the two monomer subunits. Data obtained from the publishedcrystal structure of IL-10 indicates that the functional dimer exhibitscertain similarities to IFN-γ (Zdanov et al, (1995) Structure (Lond)3:591-601). The description herein generally refers to the homodimer;however, certain aspects of the discussion can also apply to a monomer,as will be apparent from the context.

The various embodiments of the present disclosure contemplate humanIL-10 (NP_000563) and murine IL-10 (NP_034678), which exhibit 80%homology, and use thereof. In addition, the scope of the presentdisclosure includes IL-10 orthologs, and modified forms thereof, fromother mammalian species, including rat (accession NP_036986.2; GI148747382); cow (accession NP_776513.1; GI 41386772); sheep (accessionNP_001009327.1; GI 57164347); dog (accession ABY86619.1; GI 166244598);and rabbit (accession AAC23839.1; GI 3242896).

As alluded to above, the terms “IL-10”, “IL-10 polypeptide(s), “IL-10molecule(s)”, “IL-10 agent(s)” and the like are intended to be broadlyconstrued and include, for example, human and non-human IL-10-relatedpolypeptides, including homologs, variants (including muteins), andfragments thereof, as well as IL-10 polypeptides having, for example, aleader sequence (e.g., the signal peptide), and modified versions of theforegoing. In further particular embodiments, IL-10, IL-10polypeptide(s), and IL-10 agent(s) are agonists.

The IL-10 receptor, a type II cytokine receptor, consists of alpha andbeta subunits, which are also referred to as R1 and R2, respectively.Receptor activation requires binding to both alpha and beta. Onehomodimer of an IL-10 polypeptide binds to alpha and the other homodimerof the same IL-10 polypeptide binds to beta.

The utility of recombinant human IL-10 is frequently limited by itsrelatively short serum half-life, which can be due to, for example,renal clearance, proteolytic degradation and monomerization in the bloodstream. As a result, various approaches have been explored to improvethe pharmacokinetic profile of IL-10 without disrupting its dimericstructure and thus adversely affecting its activity. Pegylation of IL-10results in improvement of certain pharmacokinetic parameters (e.g.,serum half-life) and/or enhancement of activity. As used herein, theterms “pegylated IL-10” and “PEG-IL-10” refer to an IL-10 moleculehaving one or more polyethylene glycol molecules covalently attached toat least one amino acid residue of the IL-10 protein, generally via alinker, such that the attachment is stable. The terms “monopegylatedIL-10” and “mono-PEG-IL-10” indicate that one polyethylene glycolmolecule is covalently attached to a single amino acid residue on onesubunit of the IL-10 dimer, generally via a linker. As used herein, theterms “dipegylated IL-10” and “di-PEG-IL-10” indicate that at least onepolyethylene glycol molecule is attached to a single residue on eachsubunit of the IL-10 dimer, generally via a linker.

In certain embodiments, the PEG-IL-10 used in the present disclosure isa mono-PEG-IL-10 in which one to nine PEG molecules are covalentlyattached via a linker to the alpha amino group of the amino acid residueat the N-terminus of one subunit of the IL-10 dimer. Monopegylation onone IL-10 subunit generally results in a non-homogeneous mixture ofnon-pegylated, monopegylated and dipegylated IL-10 due to subunitshuffling. Moreover, allowing a pegylation reaction to proceed tocompletion will generally result in non-specific and multi-pegylatedIL-10, thus reducing its bioactivity. Thus, particular embodiments ofthe present disclosure comprise the administration of a mixture of mono-and di-pegylated IL-10 produced by the methods described herein.

In some embodiments, an N-terminal pegylation chemistry strategy can beused that results in pegylation of the N-terminus with approximately 99%specificity over a defined time period (e.g., less than 18 hours).Allowing the chemical reaction to continue beyond that time periodresults in an increase in lysine side chain pegylation. Severalpegylation approaches are described in the Experimental section.

In particular embodiments, the average molecular weight of the PEGmoiety is between about 5 kDa and about 50 kDa. Although the method orsite of PEG attachment to IL-10 is not critical, in certain embodimentsthe pegylation does not alter, or only minimally alters, the activity ofthe IL-10 agent. In certain embodiments, the increase in half-life isgreater than any decrease in biological activity. The biologicalactivity of PEG-IL-10 is typically measured by assessing the levels ofinflammatory cytokines (e.g., TNF-β or IFN-γ) in the serum of subjectschallenged with a bacterial antigen (lipopolysaccharide (LPS)) andtreated with PEG-IL-10, as described in U.S. Pat. No. 7,052,686.

IL-10 variants (unmodified by, e.g., pegylation or HSA conjugation) canbe prepared with various objectives in mind, including increasing serumhalf-life, reducing an immune response against the IL-10, facilitatingpurification or preparation, decreasing conversion of IL-10 into itsmonomeric subunits, improving therapeutic efficacy, and lessening theseverity or occurrence of side effects during therapeutic use. The aminoacid sequence variants are usually predetermined variants not found innature, although some can be post-translational variants, e.g.,glycosylated variants. Any variant of IL-10 can be used provided itretains a suitable level of IL-10 activity. As with wild-type IL-10,these IL-10 variants can be modified (by, e.g., pegylation or Fc fusion)as described herein.

The phrase “conservative amino acid substitution” refers tosubstitutions that preserve the activity of the protein by replacing anamino acid(s) in the protein with an amino acid with a side chain ofsimilar acidity, basicity, charge, polarity, or size of the side chain.Conservative amino acid substitutions generally entail substitution ofamino acid residues within the following groups: 1) L, I, M, V, F; 2) R,K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E. Guidance forsubstitutions, insertions, or deletions can be based on alignments ofamino acid sequences of different variant proteins or proteins fromdifferent species. Thus, in addition to any naturally-occurring IL-10polypeptide, the present disclosure contemplates having 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 usually no more than 20, 10, or 5 amino acidsubstitutions, where the substitution is usually a conservative aminoacid substitution.

The present disclosure also contemplates active fragments (e.g.,subsequences) of mature IL-10 containing contiguous amino acid residuesderived from the mature IL-10. The length of contiguous amino acidresidues of a peptide or a polypeptide subsequence varies depending onthe specific naturally-occurring amino acid sequence from which thesubsequence is derived. In general, peptides and polypeptides can befrom about 20 amino acids to about 40 amino acids, from about 40 aminoacids to about 60 amino acids, from about 60 amino acids to about 80amino acids, from about 80 amino acids to about 100 amino acids, fromabout 100 amino acids to about 120 amino acids, from about 120 aminoacids to about 140 amino acids, from about 140 amino acids to about 150amino acids, from about 150 amino acids to about 155 amino acids, fromabout 155 amino acids up to the full-length peptide or polypeptide.

Additionally, IL-10 polypeptides can have a defined sequence identitycompared to a reference sequence over a defined length of contiguousamino acids (e.g., a “comparison window”). Methods of alignment ofsequences for comparison are well-known in the art. Optimal alignment ofsequences for comparison can be conducted, e.g., by the local homologyalgorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by thehomology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443(1970), by the search for similarity method of Pearson & Lipman, Proc.Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations ofthese algorithms (GAP, BESTFIT, FASTA, and TFASTA in the WisconsinGenetics Software Package, Madison, Wis.), or by manual alignment andvisual inspection (see, e.g., Current Protocols in Molecular Biology(Ausubel et al., eds. 1995 supplement)).

As an example, a suitable IL-10 polypeptide can comprise an amino acidsequence having at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 95%, at least about 98%, or atleast about 99%, amino acid sequence identity to a contiguous stretch offrom about 20 amino acids to about 40 amino acids, from about 40 aminoacids to about 60 amino acids, from about 60 amino acids to about 80amino acids, from about 80 amino acids to about 100 amino acids, fromabout 100 amino acids to about 120 amino acids, from about 120 aminoacids to about 140 amino acids, from about 140 amino acids to about 150amino acids, from about 150 amino acids to about 155 amino acids, fromabout 155 amino acids up to the full-length peptide or polypeptide.

As discussed further below, the IL-10 polypeptides can be isolated froma non-natural source (e.g., an environment other than itsnaturally-occurring environment) and can also be recombinantly made(e.g., in a genetically modified host cell such as bacteria, yeast,Pichia, insect cells, and the like), where the genetically modified hostcell is modified with a nucleic acid comprising a nucleotide sequenceencoding the polypeptide. The IL-10 polypeptides can also besynthetically produced (e.g., by cell-free chemical synthesis).

Nucleic acid molecules encoding the IL-10 agents are contemplated by thepresent disclosure, including their naturally-occurring andnon-naturally occurring isoforms, allelic variants and splice variants.The present disclosure also encompasses nucleic acid sequences that varyin one or more bases from a naturally-occurring DNA sequence but stilltranslate into an amino acid sequence that corresponds to an IL-10polypeptide due to degeneracy of the genetic code.

As previously indicated, the present disclosure also contemplates theuse of gene therapy in conjunction with the teachings herein. Genetherapy is effected by delivering genetic material, usually packaged ina vector, to endogenous cells within a subject in order to introducenovel genes, to introduce additional copies of pre-existing genes, toimpair the functioning of existing genes, or to repair existing butnon-functioning genes. Once inside cells, the nucleic acid is expressedby the cell machinery, resulting in the production of the protein ofinterest. In the context of the present disclosure, gene therapy is usedas a therapeutic to deliver nucleic acid that encodes an IL-10 agent foruse in the treatment or prevention of a disease, disorder or conditiondescribed herein.

As alluded to above, for gene therapy uses and methods, a cell in asubject can be transformed with a nucleic acid that encodes anIL-10-related polypeptide as set forth herein in vivo. Alternatively, acell can be transformed in vitro with a transgene or polynucleotide, andthen transplanted into a tissue of a subject in order to effecttreatment. In addition, a primary cell isolate or an established cellline can be transformed with a transgene or polynucleotide that encodesan IL-10-related polypeptide, and then optionally transplanted into atissue of a subject.

Cholesterol Homeostasis

Physiology:

Cholesterol plays an indispensable role in a vast array of physiologicalprocesses, including cell membrane structure, and biosynthesis ofsteroid hormones, bile acids and vitamin D. Cholesterol synthesisentails a complex 37-step process that begins with the reduction of3-hydroxy-3-methylglutaryl CoA (HMG-CoA) to mevalonate by the enzymeHGM-CoA reductase. This is the regulated, rate-limiting and irreversiblestep in cholesterol synthesis and is the site of action for the statindrugs (HMG-CoA reductase competitive inhibitors).

The liver is the major regulator of cholesterol. Not only is it the siteof formation of VLDL, the precursor of most LDL in the circulation, itis also the location where the vast majority of receptor-mediatedclearance of LDL takes place.

The liver initially clears all the cholesterol that is absorbed from thesmall intestine. Absorption of excess cholesterol may increase theamount of cholesterol stored in the liver, resulting in increased VLDLsecretion (and thus LDL formation) and down-regulation of hepaticLDL-receptor activity. On average, about half of all cholesterolentering the intestine is absorbed. The fractional absorption ratevaries greatly among individuals, which may explain, at least in part,why some patients respond poorly, or not at all, to statins and otherclasses of lipid-lowering drugs. See, e.g., Turley, S D, (2004) Clin.Cardiol. 6 Suppl 3:11116-21. The liver also recycles cholesterol byexcreting it in a non-esterified form (via bile) into the digestivetract.

Lipid Panel:

Total cholesterol is defined as the sum of LDL, HDL, and VLDL. Ingeneral, total blood cholesterol levels <200 mg/dL are considerednormal, levels between 200-239 mg/dL are considered borderline-high, andlevels >240 mg/dL are considered high.

Since 1988, the National Cholesterol Education Program (NCEP) has issuedguidelines identifying LDL as the primary target of cholesterol therapy.The current guidelines, set forth in Adult Treatment Panel-III(ATP-III), set a goal for LDL<100 mg/dL (2.6 mmol/L). Increased LDL isassociated with atherosclerotic disease, which confers high risk forcoronary heart disease (CHD)-related events, including clinical CHD,symptomatic carotid artery disease, peripheral arterial disease, andabdominal aortic aneurysm. Diseases, disorders and conditions associatedwith elevated cholesterol levels, and the treatment and/or preventionthereof, are described in detail hereafter.

There is considerable evidence indicating that low levels ofhigh-density cholesterol (HDL-C, or simply HDL) are a contributoryfactor in the development of atherosclerosis and CHD. Low HDL is one ofthe most common lipid disorders in patients with premature coronaryartery disease. Patients with hypertriglyceridemia usually have lowerHDL cholesterol. Certain medications, including beta-blockers,progesterone and testosterone, also lower HDL levels.

In the average man, HDL cholesterol levels range from 40 to 50 mg/dL,whereas in the average woman, they range from 50 to 60 mg/dL. Studieshave indicated that the median values of HDL associated with the lowestrisk for atherosclerotic events are 62 mg/dL in men and 81 mg/dL inwomen. The ATP-III guidelines for lipid-lowing therapy established anHDL level below 40 mg/dL as a major positive risk factor and LDL level≥60 mg/dL as a negative risk factor (i.e., protective). A ratio of totalcholesterol to HDL of less than 5:1 is considered to be desirable.

Triglycerides are predominantly carried in the blood stream by very lowdensity lipoproteins (VLDL). There is considerable heterogeneity oftriglyceride-rich particles. Triglyceride-rich particles derived fromdietary fat—chylomicrons—are not themselves associated with CHD, but,when very high (>1,000 mg/dL) can cause pancreatitis, venous andarterial thrombi, acute heart attack and stroke. However, thesechylomicron particles are gradually reduced in size by lipoproteinlipase to intermediate density lipoproteins (IDL) which are atherogenic.Similarly, VLDL from the liver is reduced in size by lipoprotein lipase,producing atherogenic IDL. VLDL is predictive of progression of coronaryartery disease and CHD events, and thus hypertriglyceridemia has beenincreasingly recognized as a risk factor for CHD.

High triglyceride levels either result from genetic causes or areacquired. In terms of genetic causes, about 1/500 people have aninherited tendency towards high plasma triglycerides. Acquired hightriglycerides are most commonly associated with excessive alcoholintake, exogenous estrogens or estrogen agonists, poorly controlleddiabetes, beta-blockers, corticosteroids, and uremia. Triglycerideslevels in excess of 1,000 mg/dL reflect an acquired cause for hightriglycerides superimposed on a genetic cause. Less common causes ofacquired high triglycerides include kidney failure, nephrotic syndrome,albuminuria, hypothyroidism, many liver diseases, hemochromatosis,hyperparathyroidism, and glycogen storage disease.

According to the American Heart Association, triglyceride levels of lessthan 150 mg/dL are normal; levels from 150 to 199 mg/dL are borderlinehigh; levels from 200 to 499 mg/dL are high; and levels ≥500 mg/dL arevery high. In general, triglyceride levels between 150 and 200 mg/dL arenot pharmacologically treated.

Testing:

Several general methods and systems have been used in evaluating asubject's lipid profile. Any method or system, now in existence orsubsequently developed, may be used in conjunction with the teachings ofthe present disclosure.

Fasting cholesterol tests, which generally utilize a colorimetric assaysystem, are the traditional means for measuring total serum cholesterol.Such tests require blood to be drawn after a 12-hour fast to determine alipoprotein profile. Usually, only the total cholesterol, HDL, andtriglycerides are measured; for cost reasons, VLDL is generally notmeasured, but rather is estimated as one-fifth of the triglycerides, andthe LDL is estimated using the Friedewald formula. Although such testsare inexpensive and widely available (e.g., Sigma-Aldrich, St. Louis,Mo.; BioVision, Inc., Milpitas, Calif.), they require fasting and arenot as sensitive as other tests because LDL is estimated rather thandetermined accurately.

When assessing hypercholesterolemia, it is frequently useful to measureall lipoprotein subfractions (VLDL, IDL, LDL and HDL). Because aparticular therapeutic goal is to decrease LDL (while maintaining orincreasing HDL), cholesterol tests that directly measure LDL levels aremore accurate, and they are especially useful for those patients whohave elevated triglycerides. Though commercially available (e.g.,Beckman Coulter, Inc.; Brea, Calif.), use of these direct measurementtests is sometimes limited due to their cost.

The Role of Kupffer Cells in Cholesterol Homeostasis

Macrophages are often categorized by function and location; bloodmonocytes, liver Kupffer cells (liver-specific macrophages), fixedtissue macrophages, and various dendritic cells are among the mostprevalent types of macrophages.

Kupffer cells (KCs), large fixed macrophages constituting 80-90% of thetissue macrophages present in the body, reside within the lumen of thehepatic sinusoids and exhibit endocytic activity against blood-bornematerials entering the liver. KCs play a major role in the physiologicalmaintenance of the hepatic architecture and are intimately involved inthe liver's response to infection (e.g., HCV), toxins (e.g., alcohol anddrugs), ischemia, resection and other stresses (e.g., trauma).

Upon activation (by, for example, bacterial endotoxins), KCs releasevarious factors, including cytokines, prostanoides, nitric oxide andreactive oxygen species, that regulate the phenotype of KCs themselves,and the phenotypes of neighboring cells such as hepatocytes, stellatecells, endothelial cells and other immune cells that traffic through theliver. Macrophage scavenger receptors are expressed in KCs, and suchscavenger receptors are involved not only in bactericidal processes, butalso in lipid metabolism. Evidence suggests that KCs represent adistinct cell population with unique differentiation mechanisms,metabolic functions, and responsiveness to inflammatory agents.

STAT3.

The transcription factor STAT3 (signal transducer and activator oftranscription 3) is a member of the STAT protein family. In response tocytokines and growth factors, STAT family members are phosphorylated byreceptor-associated kinases; thereafter, they form homo- or heterodimersthat translocate to the cell nucleus, where they act as transcriptionactivators. STAT3 plays a key role in many cellular processes, includingcell growth and apoptosis. It is essential for the differentiation ofTH17 helper T cells, which play a role in a variety of autoimmunediseases. Moreover, STAT3 has been implicated in the regulation of lipidmetabolism. (See Kinoshita et al., Kobe J. Med. Sci., Vol. 54, No. 4,pp. E200-E208, 2008).

STAT3 phosphorylation occurs in response to various cytokines and growthfactors, including certain interleukins (e.g., IL-5, IL-6 and IL-10),leukemia inhibitory factor (LIF), epidermal growth factor (EGF), certaininterferons, hepatocyte growth factor (HGF), bone morphogenetic protein2 (BMP-2), the cytokine oncostatin M (OSM), and the hormone leptin. IL-6lowers serum cholesterol in mice and humans, human LIF decreases serumcholesterol in hypercholesterolemic rabbits, and OSM decreases serumcholesterol in hypercholesterolemic hamsters; this cholesterol reductionis believed to be effected through upregulation of the LDL receptor.

The present disclosure is based, in part, on the discovery that thecapacity of KC to remove lipoproteins from the serum can be modulated(e.g., increased) in order to effect desirable metabolic effects (e.g.,cholesterol lowering). In particular aspects, the present disclosure isdrawn to methods of identifying agents that induce the phosphorylationof STAT3 in KCs, thereby increasing their ability to remove lipoproteinsfrom the serum. As noted herein, while an understanding of theunderlying mechanism of action by which KCs are involved in cholesterollowering is not required in order to practice the present disclosure,agents that induce STAT3 phosphorylation in KCs are believed to increaseKCs' capacity for scavenging lipoproteins from the serum. In thiscontext, the phrase “agents that induce STAT3 phosphorylation” is meantto refer broadly to any molecule (e.g., a small molecule, a polypeptide,and an antibody) that causes, directly or indirectly, in whole or inpart, an increase in phosphorylated STAT3. In particular embodiments,such agents are factors which drive the phosphorylation of STAT3 (e.g.,IL-10, IL-6, LIF and Oncostatin M).

Scavenger Receptors.

Scavenger receptors participate in the removal of many foreignsubstances and waste materials in the body by extensive ligandspecificity and a variety of receptor molecules. They constitute a groupof receptors that recognize and uptake negatively chargedmacromolecules, as well as LDL that has been modified by oxidation(oxLDL) or acetylation (acLDL).

Scavenger receptors are generally categorized into threecategories—Class A, Class B, and Class C—according to their structuralcharacteristics. Class A Scavenger Receptors (Scavenger receptors type 1(SR-A1) and 2 (SR-A2)) are trimers that preferentially bind modified LDL(e.g., oxLDL and acLDL) and have a collagen-like domain essential forligand binding. Class A members include MSR1 (also known as SCARA1),MARCO (also known as SCARA2), SCARA3, COLEC12 (also known as SCARA4),and SCARA5.

Class B Scavenger Receptors are identified as oxidized LDL receptors,and its members include CD36 and Scavenger Receptor Class BI (SR-BI).Class B Scavenger Receptors are often referred to as SCARB1 (which alsointeracts with HDL); SCARB2; and SCARB3 (also known as CD36), which hasbeen implicated in phagocytosis of apoptotic cells and in metabolism oflong-chain fatty acids. Class C Scavenger Receptors include otherreceptors that can bind to oxidized LDL, including CD68, Mucin, andLectin-like oxidized LDL receptor-1 (LOX-1).

As described herein, the uptake process of modified LDL by scavengerreceptors is followed by intracellular degradation and/or efflux ontoHLD particles. IL-10 has been shown to be involved in the uptake andefflux processes, and likely contributes to the degradation process aswell.

Scavenger receptor function was evaluated herein (see Experimentalsection) for, among others, MSR1, MARCO, SCARB1, SCARB2, and CD36. Whenthe effect of IL-10 (PEG-rMuIL-10) on the expression of genes associatedwith liver function and cholesterol regulation was assessed, only twoprimary groups of genes were altered, one of which was scavengerreceptors (see Example 2 and FIG. 2). As indicated in FIGS. 2E-H, Msr1and Marco, Type A scavenger receptors, were induced by 2-7 fold inwild-type and LDLR−/− mice on normal chow diet; and wild-type andLDLR−/− mice on high-fat chow diet. In addition, because scavengerreceptors are predominantly expressed on macrophage-type cells,differences in gene expression of F4/80 and CD14, two cell surfaceproteins most often expressed on liver tissue resident macrophages, wereassessed, and these genes were moderately induced across the differentgenetic backgrounds and dietary conditions (see FIGS. 2E-H). FIGS. 3A-Dindicate the effect of PEG-rMuIL-10 on Msr1 (see Example 3). These dataconfirm the role of scavenger receptors in aspects of liver function andcholesterol regulation.

Effect of PEG-IL-10 and Other Cytokines on Cholesterol Homeostasis andKC Function

IL-10's Role in Cholesterol Homeostasis.

The extent by which PEG-IL-10 reduces plasma cholesterol is related tothe subject's total cholesterol level. This was observed in both miceand humans. As indicated in Example 1 and FIG. 1, PEG-rMuIL-10 loweredplasma cholesterol only in hypercholesterolemic mice, and PEG-rHuIL-10lowered plasma cholesterol only in patients with elevated plasmacholesterol levels. To illustrate, cancer patients with border-line high(˜200 mg/dL) total cholesterol achieved an approximately 40% cholesterolreduction following administration of PEG-rHuIL-10 SC daily, whereaspatients with low (˜100 mg/dL) total cholesterol were unaffected. Thesedata suggest that PEG-IL-10 is more efficacious in patient populationsthat would benefit the most from cholesterol reduction.

This response is believed to be triggered by the level of IL-10Rαexpression, as a high fat diet induced upregulation of the IL-10Rα inmouse liver (data not shown).

The present disclosure contemplates the administration of cytokines(e.g., PEG-IL-10) to subjects that would benefit from cholesterolreduction, regardless of their total cholesterol levels. Thus, forexample, in some embodiments PEG-IL-10 is administered to subjectshaving a total cholesterol level of at least 150 mg/dL, at least 160mg/dL, at least 170 mg/dL, at least 180 mg/dL, at least 190 mg/dL, atleast 200 mg/dL, at least 210 mg/dL, at least 220 mg/dL, at least 230mg/dL, at least 240 mg/dL, at least 250 mg/dL, at least 260 mg/dL, atleast 270 mg/dL, at least 280 mg/dL, at least 290 mg/dL, or at least 300mg/dL. In other embodiments, PEG-IL-10 is administered to subjectshaving a total cholesterol level of at least 325 mg/dL, at least 350mg/dL, at least 375 mg/dL, at least 400 mg/dL, at least 425 mg/dL, atleast 450 mg/dL, at least 475 mg/dL, or at least 500 mg/dL.

In particular embodiments, an IL-10 agent (or other cytokine agent)disclosed herein (e.g., PEG-IL-10) has an anti-hyperlipidemia activitycapable of reducing the levels of VLDL, IDL, LDL, or a combinationthereof by, e.g., at least 10%, at least 15%, at least 20%, at least25%, at least 30%, at least 35%, at least 40%, at least 45%, at least50%, at least 55%, at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 90% or at least 95%. In yetother embodiments, an IL-10 agent (or other cytokine agent) disclosedherein (e.g., PEG-IL-10) has anti-hyperlipidemia activity capable ofreducing the levels of VLDL, IDL, LDL, or a combination thereof in arange from, e.g., about 10% to about 100%, about 20% to about 100%,about 30% to about 100%, about 40% to about 100%, about 50% to about100%, about 60% to about 100%, about 70% to about 100%, or about 80% toabout 100%; about 10% to about 90%, about 20% to about 90%, about 30% toabout 90%, about 40% to about 90%, about 50% to about 90%, about 60% toabout 90%, or about 70% to about 90%; about 10% to about 80%, about 20%to about 80%, about 30% to about 80%, about 40% to about 80%, about 50%to about 80%, or about 60% to about 80%; about 10% to about 70%, about20% to about 70%, about 30% to about 70%, about 40% to about 70%, orabout 50% to about 70%.

In another embodiment of the present disclosure, an IL-10 agent (orother cytokine agent) disclosed herein (e.g., PEG-IL-10) increases thelevel of HDL. In some other (often more frequent) embodiments, the levelof HDL itself does not increase; rather the levels of both LDL and HDLdecrease, but the level of LDL decrease exceeds the level of HDLdecrease such that the change in the final ratio of LDL to HDL isconsistent with the HDL lipid hypothesis. In an aspect of theseembodiments, the IL-10 agent (or other cytokine agent) increases thelevel of HDL relative to LDL by, e.g., at least 2%, at least 3%, atleast 10%, at least 12%, at least 15%, at least 17%, at least 20%, atleast 22%, at least 25%, at least 27%, at least 30%, at least 32%, atleast 35%, at least 37%, at least 40%, at least 42%, at least 45% or atleast 47%. In yet other aspects of these embodiments, the IL-10 agentincreases the level of HDL relative to LDL in a range from, e.g., about2% to about 100%; about 10% to about 50%, about 15% to about 50%, about20% to about 50%, about 25% to about 50%, about 30% to about 50%, about35% to about 50%, or about 40% to about 50%; about 2% to about 45%,about 10% to about 45%, about 15% to about 45%, about 20% to about 45%,about 25% to about 45%, about 30% to about 45%, or about 35% to about45%; about 2% to about 40%, about 10% to about 40%, about 15% to about40%, about 20% to about 40%, about 25% to about 40%, or about 30% toabout 40%; about 2% to about 35%, about 10% to about 35%, about 15% toabout 35%, about 20% to about 35%, or about 25% to about 35%.

IL-10's Role in KC Function.

Although an understanding of the underlying mechanism by which IL-10exerts its effects is not required in order to practice the presentdisclosure, as previously alluded to, IL-10 activates the myeloid immunesystem through activation of liver resident KCs to dramatically reducesystemic cholesterol levels in subjects having hypercholesterol.

As detailed in the Experimental section, the regulatory effect ofPEG-rHuIL-10 on total plasma cholesterol was determined using a murinesurrogate, PEG-rMuIL-10. FIGS. 2A-D indicate that PEG-rMuIL-10 decreasedtwo genes (Hmgcs1 and Hmgcs2) involved in cholesterol synthesis.

Administration of PEG-rMuIL-10 to LDLR−/− mice decreased total plasmacholesterol in a manner that is commensurate with increased scavengerreceptor expression in KCs, and KCs were shown to be the predominantmyeloid cell type that responded to PEG-rMuIL-10 with increasedscavenging of LDL. Because removal of all phagocytotic cells, themajority of which are myeloid lineage cells, dramatically increasedplasma cholesterol, phagocytotic cell populations play a major role inthe regulation of plasma cholesterol levels.

Treatment of NAFLD and NASH with IL-10 and Other Cytokines

Non-alcoholic steatohepatitis (NASH), considered part of a spectrum ofnon-alcoholic fatty liver diseases (NAFLD), causes inflammation andaccumulation of fat and fibrous tissue in the liver. Although the exactcause of NASH is unknown, risk factors include central obesity, type-2diabetes mellitus, insulin resistance (IR) and dyslipidemia;combinations of the foregoing are frequently described as the metabolicsyndrome. In addition, certain drugs have been linked to NASH, includingtamoxifen, amiodarone and steroids (e.g., prednisone andhydrocortisone). Non-alcoholic fatty liver disease is the most commoncause of chronic liver disease in the United States, and the estimatedprevalence of NAFLD is 20-30% and for NASH it is estimated at 3.5-5%.(See, e.g., Abrams, G. A., et al., Hepatology, 2004. 40(2):475-83;Moreira, R. K., Arch Pathol Lab Med, 2007. 131(11):1728-34).

NASH frequently presents with no overt symptoms, complicating itsdiagnosis. Liver function tests generally begin the diagnostic process,with levels of AST (aspartate aminotransferase) and ALT (alanineaminotransferase) elevated in about 90% percent of individuals withNASH. Other blood tests are often used for ruling out other causes ofliver disease, such as hepatitis. Imaging tests (e.g., ultrasound, CTscan, or MRI) may reveal fat accumulation in the liver but frequentlycannot differentiate NASH from other causes of liver disease that have asimilar appearance. A liver biopsy is required to confirm NASH.

The prognosis for individuals suffering from NASH is difficult topredict, although features in the liver biopsy can be helpful. The mostserious complication of NASH is cirrhosis, which occurs when the liverbecomes severely scarred. It has been reported that between 8 and 26percent of individuals with NASH develop cirrhosis, and it is predictedthat NASH will be the leading indication for liver transplantation by2020.

At the present time, treatment of NASH focuses primarily onpharmacological and non-pharmacological management of those medicalconditions associated with it, including hyperlipidemia, diabetes andobesity. Although not curative, pharmacological intervention of NASHitself includes treatment with vitamin E, pioglitazone, metformin,statins, omega-3 fatty acids, and ursodeoxycholic acid (UDCA(ursodiol)). Other agents being evaluated, currently approved fordifferent indications, include losartan and telisartan, exenatide, GLP-1agonists, DPP IV inhibitors, and carbamazepine. Combination therapy ishoped to offer new opportunities for disease control.

Historically, the activation of Kupffer cells has been associated withthe initiation and progression of liver disease (Kolios, G. et al.,World J. Gastroenterol, 2006. 12(46):7413-20). In particular, Kupffercells were believed to be involved in alcoholic liver disease, (ALD)(Eguchi, H., et al., Hepatology, 1991. 13(4):751-57), non-alcoholicfatty liver disease, (NAFLD) (Stienstra R., et al., Hepatology, 2010.51(2):511-22, and non-alcoholic steatohepatitis, (NASH) (Tomita, K. etal., Gut, 2006. 55(3):415-24). In general, these diseases were thoughtto be linked by the inappropriate accumulation of liver cholesterol andtriglycerides within both the Kupffer cells and hepatocytes. Thus, therehas been the belief that depletion of KC function might betherapeutically relevant in the treatment of metabolic disorders such asNASH. (Neyrinck et al., Biochem. Biophys. Res. Comm., 385:351-56(2009)).

The teachings of the present disclosure are diametrically opposed tothis belief. While it has been reported that activation of KCs isnecessary to optimize liver regeneration (Bilzer, M et al., (2006),Liver Int, 26:1175-86), it was not previously recognized that KCs playan integral role in cholesterol and triglyceride regulation. Embodimentsof the present disclosure are associated with the novel finding thataspects of KC function entail significant uptake of serum cholesteroland, thereafter, its removal and catabolism. The alteration to collagendeposition described herein is linked to the removal of triglycerides,which then leads to the removal of the pro-inflammatory stimulus,permitting the resolution of peri-portal injury and fibrosis.

As described in the Experimental section, IL-10 (e.g., PEG-IL-10)activation of KCs' scavenging capacity is associated with decreases inthe accumulation of liver cholesterol and triglycerides, and certainembodiments of the present disclosure contemplate the use of PEG-IL-10to induce the removal of accumulated liver triglycerides andcholesterol. Induction of the removal of accumulated liver triglyceridesand cholesterol, in turn, results in the reversal of early liverfibrosis and facilitates the restoration of liver health through, forexample, increasing the number of hepatocytes in the liver. These datasupport the use of IL-10 (e.g., PEG-Il-10) for the treatment of NAFLDand NASH. Thus, particular embodiments of the present disclosurecontemplate the use of IL-10 (including human and non-humanIL-10-related polypeptides, including homologs, variants (includingmuteins), and fragments thereof) in the treatment and/or prevention ofNAFLD and NASH.

In addition, other cytokines can effect depletion of KC function and canbe therapeutically relevant in the treatment of liver-related disorderssuch as NAFLD and NASH. As used herein, the term “cytokine(s)” is meantto have its ordinary meaning in the art. Cytokines are involved in cellsignaling—cells of the immune system communicate with one another byreleasing and responding to cytokines. Cytokines encompass a diversearray of proteins that include interleukins, interferons, chemokines,lymphokines, and tumor necrosis factor. They are produced by broad rangeof cells, including macrophages, B lymphocytes, T lymphocytes, mastcells, fibroblasts, and endothelial cells.

Cytokines modulate the balance between humoral- and cell-based immuneresponses and regulate the maturation, growth, and responsiveness ofparticular cell populations. They also play an integral role in hostresponses to, for example, infection, immune responses, inflammation,and trauma. Although not readily subject to definitive classification,cytokines are sometimes classified as interleukins, lymphokines,monokines, interferons, colony stimulating factors and chemokines.

In particular embodiments, the present disclosure contemplates the useof cytokines in the treatment and/or prevention of liver-relateddisorders such as NAFLD and NASH. Dosing regimens applicable to cytokineagents in general, and IL-10 agents in particular, are describedelsewhere herein.

Methods of Production of IL-10

A polypeptide of the present disclosure can be produced by any suitablemethod, including non-recombinant (e.g., chemical synthesis) andrecombinant methods.

A. Chemical Synthesis

Where a polypeptide is chemically synthesized, the synthesis may proceedvia liquid-phase or solid-phase. Solid-phase peptide synthesis (SPPS)allows the incorporation of unnatural amino acids and/or peptide/proteinbackbone modification. Various forms of SPPS, such as9-fluorenylmethoxycarbonyl (Fmoc) and t-butyloxycarbonyl (Boc), areavailable for synthesizing polypeptides of the present disclosure.Details of the chemical syntheses are known in the art (e.g., Ganesan A.(2006) Mini Rev. Med. Chem. 6:3-10; and Camarero J. A. et al., (2005)Protein Pept Lett. 12:723-8).

Solid phase peptide synthesis may be performed as described hereafter.The alpha functions (Nα) and any reactive side chains are protected withacid-labile or base-labile groups. The protective groups are stableunder the conditions for linking amide bonds but can readily be cleavedwithout impairing the peptide chain that has formed. Suitable protectivegroups for the α-amino function include, but are not limited to, thefollowing: Boc, benzyloxycarbonyl (Z), O-chlorbenzyloxycarbonyl,bi-phenylisopropyloxycarbonyl, tert-amyloxycarbonyl (Amoc), α,α-dimethyl-3,5-dimethoxy-benzyloxycarbonyl, o-nitrosulfenyl,2-cyano-t-butoxy-carbonyl, Fmoc,1-(4,4-dimethyl-2,6-dioxocylohex-1-ylidene)ethyl (Dde) and the like.

Suitable side chain protective groups include, but are not limited to:acetyl, allyl (All), allyloxycarbonyl (Alloc), benzyl (Bzl),benzyloxycarbonyl (Z), t-butyloxycarbonyl (Boc), benzyloxymethyl (Bom),o-bromobenzyloxycarbonyl, t-butyl (tBu), t-butyldimethylsilyl,2-chlorobenzyl, 2-chlorobenzyloxycarbonyl, 2,6-dichlorobenzyl,cyclohexyl, cyclopentyl,1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl (Dde), isopropyl,4-methoxy-2,3-6-trimethylbenzylsulfonyl (Mtr),2,3,5,7,8-pentamethylchroman-6-sulfonyl (Pmc), pivalyl,tetrahydropyran-2-yl, tosyl (Tos), 2,4,6-trimethoxybenzyl,trimethylsilyl and trityl (Trt).

In the solid phase synthesis, the C-terminal amino acid is coupled to asuitable support material. Suitable support materials are those whichare inert towards the reagents and reaction conditions for the step-wisecondensation and cleavage reactions of the synthesis process and whichdo not dissolve in the reaction media being used. Examples ofcommercially-available support materials include styrene/divinylbenzenecopolymers which have been modified with reactive groups and/orpolyethylene glycol; chloromethylated styrene/divinylbenzene copolymers;hydroxymethylated or aminomethylated styrene/divinylbenzene copolymers;and the like. When preparation of the peptidic acid is desired,polystyrene (1%)-divinylbenzene or TentaGel® derivatized with4-benzyloxybenzyl-alcohol (Wang-anchor) or 2-chlorotrityl chloride canbe used. In the case of the peptide amide, polystyrene (1%)divinylbenzene or TentaGel® derivatized with5-(4′-aminomethyl)-3′,5′-dimethoxyphenoxy)valeric acid (PAL-anchor) orp-(2,4-dimethoxyphenyl-amino methyl)-phenoxy group (Rink amide anchor)can be used.

The linkage to the polymeric support can be achieved by reacting theC-terminal Fmoc-protected amino acid with the support material by theaddition of an activation reagent in ethanol, acetonitrile,N,N-dimethylformamide (DMF), dichloromethane, tetrahydrofuran,N-methylpyrrolidone or similar solvents at room temperature or elevatedtemperatures (e.g., between 40° C. and 60° C.) and with reaction timesof, e.g., 2 to 72 hours.

The coupling of the Nα-protected amino acid (e.g., the Fmoc amino acid)to the PAL, Wang or Rink anchor can, for example, be carried out withthe aid of coupling reagents such as N,N′-dicyclohexylcarbodiimide(DCC), N,N′-diisopropylcarbodiimide (DIC) or other carbodiimides,2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate(TBTU) or other uronium salts, O-acyl-ureas,benzotriazol-1-yl-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP) or other phosphonium salts, N-hydroxysuccinimides, otherN-hydroxyimides or oximes in the presence or absence of1-hydroxybenzotriazole or 1-hydroxy-7-azabenzotriazole, e.g., with theaid of TBTU with addition of HOBt, with or without the addition of abase such as, for example, diisopropylethylamine (DIEA), triethylamineor N-methylmorpholine, e.g., diisopropylethylamine with reaction timesof 2 to 72 hours (e.g., 3 hours in a 1.5 to 3-fold excess of the aminoacid and the coupling reagents, for example, in a 2-fold excess and attemperatures between about 10° C. and 50° C., for example, 25° C. in asolvent such as dimethylformamide, N-methylpyrrolidone ordichloromethane, e.g., dimethylformamide).

Instead of the coupling reagents, it is also possible to use the activeesters (e.g., pentafluorophenyl, p-nitrophenyl or the like), thesymmetric anhydride of the Nα-Fmoc-amino acid, its acid chloride or acidfluoride, under the conditions described above.

The Nα-protected amino acid (e.g., the Fmoc amino acid) can be coupledto the 2-chlorotrityl resin in dichloromethane with the addition of DIEAand having reaction times of 10 to 120 minutes, e.g., 20 minutes, but isnot limited to the use of this solvent and this base.

The successive coupling of the protected amino acids can be carried outaccording to conventional methods in peptide synthesis, typically in anautomated peptide synthesizer. After cleavage of the Nα-Fmoc protectivegroup of the coupled amino acid on the solid phase by treatment with,e.g., piperidine (10% to 50%) in dimethylformamide for 5 to 20 minutes,e.g., 2×2 minutes with 50% piperidine in DMF and 1×15 minutes with 20%piperidine in DMF, the next protected amino acid in a 3 to 10-foldexcess, e.g., in a 10-fold excess, is coupled to the previous amino acidin an inert, non-aqueous, polar solvent such as dichloromethane, DMF ormixtures of the two and at temperatures between about 10° C. and 50° C.,e.g., at 25° C. The previously mentioned reagents for coupling the firstNα-Fmoc amino acid to the PAL, Wang or Rink anchor are suitable ascoupling reagents. Active esters of the protected amino acid, orchlorides or fluorides or symmetric anhydrides thereof can also be usedas an alternative.

At the end of the solid phase synthesis, the peptide is cleaved from thesupport material while simultaneously cleaving the side chain protectinggroups. Cleavage can be carried out with trifluoroacetic acid or otherstrongly acidic media with addition of 5%-20% V/V of scavengers such asdimethylsulfide, ethylmethylsulfide, thioanisole, thiocresol, m-cresol,anisole ethanedithiol, phenol or water, e.g., 15% v/vdimethylsulfide/ethanedithiol/m-cresol 1:1:1, within 0.5 to 3 hours,e.g., 2 hours. Peptides with fully protected side chains are obtained bycleaving the 2-chlorotrityl anchor with glacial aceticacid/trifluoroethanol/dichloromethane 2:2:6. The protected peptide canbe purified by chromatography on silica gel. If the peptide is linked tothe solid phase via the Wang anchor and if it is intended to obtain apeptide with a C-terminal alkylamidation, the cleavage can be carriedout by aminolysis with an alkylamine or fluoroalkylamine. The aminolysisis carried out at temperatures between about −10° C. and 50° C. (e.g.,about 25° C.), and reaction times between about 12 and 24 hours (e.g.,about 18 hours). In addition, the peptide can be cleaved from thesupport by re-esterification, e.g., with methanol.

The acidic solution that is obtained may be admixed with a 3 to 20-foldamount of cold ether or n-hexane, e.g., a 10-fold excess of diethylether, in order to precipitate the peptide and hence to separate thescavengers and cleaved protective groups that remain in the ether. Afurther purification can be carried out by re-precipitating the peptideseveral times from glacial acetic acid. The precipitate that is obtainedcan be taken up in water or tert-butanol or mixtures of the twosolvents, e.g., a 1:1 mixture of tert-butanol/water, and freeze-dried.

The peptide obtained can be purified by various chromatographic methods,including ion exchange over a weakly basic resin in the acetate form;hydrophobic adsorption chromatography on non-derivatizedpolystyrene/divinylbenzene copolymers (e.g., Amberlite® XAD); adsorptionchromatography on silica gel; ion exchange chromatography, e.g., oncarboxymethyl cellulose; distribution chromatography, e.g., on Sephadex®G-25; countercurrent distribution chromatography; or high pressureliquid chromatography (HPLC) e.g., reversed-phase HPLC on octyl oroctadecylsilylsilica (ODS) phases.

B. Recombinant Production

Methods describing the preparation of human and mouse IL-10 can be foundin, for example, U.S. Pat. No. 5,231,012, which teaches methods for theproduction of proteins having IL-10 activity, including recombinant andother synthetic techniques. IL-10 can be of viral origin, and thecloning and expression of a viral IL-10 from Epstein Barr virus (BCRF1protein) is disclosed in Moore et al., (1990) Science 248:1230. IL-10can be obtained in a number of ways using standard techniques known inthe art, such as those described herein. Recombinant human IL-10 is alsocommercially available, e.g., from PeproTech, Inc., Rocky Hill, N.J.

Where a polypeptide is produced using recombinant techniques, thepolypeptide may be produced as an intracellular protein or as a secretedprotein, using any suitable construct and any suitable host cell, whichcan be a prokaryotic or eukaryotic cell, such as a bacterial (e.g., E.coli) or a yeast host cell, respectively. Other examples of eukaryoticcells that may be used as host cells include insect cells, mammaliancells, and/or plant cells. Where mammalian host cells are used, they mayinclude human cells (e.g., HeLa, 293, H9 and Jurkat cells); mouse cells(e.g., NIH3T3, L cells, and C127 cells); primate cells (e.g., Cos 1, Cos7 and CV1); and hamster cells (e.g., Chinese hamster ovary (CHO) cells).

A variety of host-vector systems suitable for the expression of apolypeptide may be employed according to standard procedures known inthe art. See, e.g., Sambrook et al., 1989 Current Protocols in MolecularBiology Cold Spring Harbor Press, New York; and Ausubel et al. 1995Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods forintroduction of genetic material into host cells include, for example,transformation, electroporation, conjugation, calcium phosphate methodsand the like. The method for transfer can be selected so as to providefor stable expression of the introduced polypeptide-encoding nucleicacid. The polypeptide-encoding nucleic acid can be provided as aninheritable episomal element (e.g., a plasmid) or can be genomicallyintegrated. A variety of appropriate vectors for use in production of apolypeptide of interest are commercially available.

Vectors can provide for extrachromosomal maintenance in a host cell orcan provide for integration into the host cell genome. The expressionvector provides transcriptional and translational regulatory sequences,and may provide for inducible or constitutive expression where thecoding region is operably-linked under the transcriptional control ofthe transcriptional initiation region, and a transcriptional andtranslational termination region. In general, the transcriptional andtranslational regulatory sequences may include, but are not limited to,promoter sequences, ribosomal binding sites, transcriptional start andstop sequences, translational start and stop sequences, and enhancer oractivator sequences. Promoters can be either constitutive or inducible,and can be a strong constitutive promoter (e.g., T7).

Expression constructs generally have convenient restriction siteslocated near the promoter sequence to provide for the insertion ofnucleic acid sequences encoding proteins of interest. A selectablemarker operative in the expression host may be present to facilitateselection of cells containing the vector. Moreover, the expressionconstruct may include additional elements. For example, the expressionvector may have one or two replication systems, thus allowing it to bemaintained in organisms, for example, in mammalian or insect cells forexpression and in a prokaryotic host for cloning and amplification. Inaddition, the expression construct may contain a selectable marker geneto allow the selection of transformed host cells. Selectable genes arewell known in the art and will vary with the host cell used.

Isolation and purification of a protein can be accomplished according tomethods known in the art. For example, a protein can be isolated from alysate of cells genetically modified to express the proteinconstitutively and/or upon induction, or from a synthetic reactionmixture by immunoaffinity purification, which generally involvescontacting the sample with an anti-protein antibody, washing to removenon-specifically bound material, and eluting the specifically boundprotein. The isolated protein can be further purified by dialysis andother methods normally employed in protein purification. In oneembodiment, the protein may be isolated using metal chelatechromatography methods. Proteins may contain modifications to facilitateisolation.

The polypeptides may be prepared in substantially pure or isolated form(e.g., free from other polypeptides). The polypeptides can be present ina composition that is enriched for the polypeptide relative to othercomponents that may be present (e.g., other polypeptides or other hostcell components). For example, purified polypeptide may be provided suchthat the polypeptide is present in a composition that is substantiallyfree of other expressed proteins, e.g., less than about 90%, less thanabout 60%, less than about 50%, less than about 40%, less than about30%, less than about 20%, less than about 10%, less than about 5%, orless than about 1%.

An IL-10 polypeptide may be generated using recombinant techniques tomanipulate different IL-10-related nucleic acids known in the art toprovide constructs capable of encoding the IL-10 polypeptide. It will beappreciated that, when provided a particular amino acid sequence, theordinary skilled artisan will recognize a variety of different nucleicacid molecules encoding such amino acid sequence in view of herbackground and experience in, for example, molecular biology.

Amide Bond Substitutions

In some cases, IL-10 includes one or more linkages other than peptidebonds, e.g., at least two adjacent amino acids are joined via a linkageother than an amide bond. For example, in order to reduce or eliminateundesired proteolysis or other means of degradation, and/or to increaseserum stability, and/or to restrict or increase conformationalflexibility, one or more amide bonds within the backbone of IL-10 can besubstituted.

In another example, one or more amide linkages (—CO—NH—) in IL-10 can bereplaced with a linkage which is an isostere of an amide linkage, suchas —CH₂NH—, —CH₂S—, —CH₂CH₂—, —CH═CH-(cis and trans), —COCH₂—,—CH(OH)CH₂— or —CH₂SO—. One or more amide linkages in IL-10 can also bereplaced by, for example, a reduced isostere pseudopeptide bond. SeeCouder et al. (1993) Int. J. Peptide Protein Res. 41:181-184. Suchreplacements and how to effect them are known to those of ordinary skillin the art.

Amino Acid Substitutions

One or more amino acid substitutions can be made in an IL-10polypeptide. The following are non-limiting examples:

a) substitution of alkyl-substituted hydrophobic amino acids, includingalanine, leucine, isoleucine, valine, norleucine, (S)-2-aminobutyricacid, (S)-cyclohexylalanine or other simple alpha-amino acidssubstituted by an aliphatic side chain from C₁-C₁₀ carbons includingbranched, cyclic and straight chain alkyl, alkenyl or alkynylsubstitutions;

b) substitution of aromatic-substituted hydrophobic amino acids,including phenylalanine, tryptophan, tyrosine, sulfotyrosine,biphenylalanine, 1-naphthylalanine, 2-naphthylalanine,2-benzothienylalanine, 3-benzothienylalanine, histidine, includingamino, alkylamino, dialkylamino, aza, halogenated (fluoro, chloro,bromo, or iodo) or alkoxy (from C₁-C₄)-substituted forms of theabove-listed aromatic amino acids, illustrative examples of which are:2-, 3- or 4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3-or 4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine, 5-amino-,5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-, or 4′-amino-,2′-, 3′-, or 4′-chloro-, 2, 3, or 4-biphenylalanine, 2′-, 3′-, or4′-methyl-, 2-, 3- or 4-biphenylalanine, and 2- or 3-pyridylalanine;

c) substitution of amino acids containing basic side chains, includingarginine, lysine, histidine, ornithine, 2,3-diaminopropionic acid,homoarginine, including alkyl, alkenyl, or aryl-substituted (from C₁-C₁₀branched, linear, or cyclic) derivatives of the previous amino acids,whether the substituent is on the heteroatoms (such as the alphanitrogen, or the distal nitrogen or nitrogens, or on the alpha carbon,in the pro-R position for example. Compounds that serve as illustrativeexamples include: N-epsilon-isopropyl-lysine,3-(4-tetrahydropyridyl)-glycine, 3-(4-tetrahydropyridyl)-alanine,N,N-gamma, gamma'-diethyl-homoarginine. Included also are compounds suchas alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic acid,alpha-methyl-histidine, alpha-methyl-ornithine where the alkyl groupoccupies the pro-R position of the alpha-carbon. Also included are theamides formed from alkyl, aromatic, heteroaromatic (where theheteroaromatic group has one or more nitrogens, oxygens or sulfur atomssingly or in combination), carboxylic acids or any of the manywell-known activated derivatives such as acid chlorides, active esters,active azolides and related derivatives, and lysine, ornithine, or2,3-diaminopropionic acid;

d) substitution of acidic amino acids, including aspartic acid, glutamicacid, homoglutamic acid, tyrosine, alkyl, aryl, arylalkyl, andheteroaryl sulfonamides of 2,4-diaminopriopionic acid, ornithine orlysine and tetrazole-substituted alkyl amino acids;

e) substitution of side chain amide residues, including asparagine,glutamine, and alkyl or aromatic substituted derivatives of asparagineor glutamine; and

f) substitution of hydroxyl-containing amino acids, including serine,threonine, homoserine, 2,3-diaminopropionic acid, and alkyl or aromaticsubstituted derivatives of serine or threonine.

In some cases, IL-10 comprises one or more naturally occurringnon-genetically encoded L-amino acids, synthetic L-amino acids, orD-enantiomers of an amino acid. For example, IL-10 can comprise onlyD-amino acids. For example, an IL-10 polypeptide can comprise one ormore of the following residues: hydroxyproline, β-alanine,o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid,m-aminomethylbenzoic acid, 2,3-diaminopropionic acid, α-aminoisobutyricacid, N-methylglycine (sarcosine), ornithine, citrulline,t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine,cyclohexylalanine, norleucine, naphthylalanine, pyridylalanine3-benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine,3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-2-thienylalanine,methionine sulfoxide, homoarginine, N-acetyl lysine, 2,4-diamino butyricacid, rho-aminophenylalanine, N-methylvaline, homocysteine, homoserine,κ-amino hexanoic acid, w-aminohexanoic acid, ω-aminoheptanoic acid,ω-aminooctanoic acid, ω-aminodecanoic acid, ω-aminotetradecanoic acid,cyclohexylalanine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid,δ-amino valeric acid, and 2,3-diaminobutyric acid.

Additional Modifications

A cysteine residue or a cysteine analog can be introduced into an IL-10polypeptide to provide for linkage to another peptide via a disulfidelinkage or to provide for cyclization of the IL-10 polypeptide. Methodsof introducing a cysteine or cysteine analog are known in the art; see,e.g., U.S. Pat. No. 8,067,532.

An IL-10 polypeptide can be cyclized. One or more cysteines or cysteineanalogs can be introduced into an IL-10 polypeptide, where theintroduced cysteine or cysteine analog can form a disulfide bond with asecond introduced cysteine or cysteine analog. Other means ofcyclization include introduction of an oxime linker or a lanthioninelinker; see, e.g., U.S. Pat. No. 8,044,175. Any combination of aminoacids (or non-amino acid moieties) that can form a cyclizing bond can beused and/or introduced. A cyclizing bond can be generated with anycombination of amino acids (or with an amino acid and —(CH2)_(n)-CO— or—(CH2)_(n)-C₆H₄—CO—) with functional groups which allow for theintroduction of a bridge. Some examples are disulfides, disulfidemimetics such as the —(CH2)_(n)-carba bridge, thioacetal, thioetherbridges (cystathionine or lanthionine) and bridges containing esters andethers. In these examples, n can be any integer, but is frequently lessthan ten.

Other modifications include, for example, an N-alkyl (or aryl)substitution (ψ[CONR]), or backbone crosslinking to construct lactamsand other cyclic structures. Other derivatives include C-terminalhydroxymethyl derivatives, o-modified derivatives (e.g., C-terminalhydroxymethyl benzyl ether), N-terminally modified derivatives includingsubstituted amides such as alkylamides and hydrazides.

In some cases, one or more L-amino acids in an IL-10 polypeptide isreplaced with one or more D-amino acids.

In some cases, an IL-10 polypeptide is a retroinverso analog (see, e.g.,Sela and Zisman (1997) FASEB J. 11:449). Retro-inverso peptide analogsare isomers of linear polypeptides in which the direction of the aminoacid sequence is reversed (retro) and the chirality, D- or L-, of one ormore amino acids therein is inverted (inverso), e.g., using D-aminoacids rather than L-amino acids.) See, e.g., Jameson et al. (1994)Nature 368:744; and Brady et al. (1994) Nature 368:692).

An IL-10 polypeptide can include a “Protein Transduction Domain” (PTD),which refers to a polypeptide, polynucleotide, carbohydrate, or organicor inorganic molecule that facilitates traversing a lipid bilayer,micelle, cell membrane, organelle membrane, or vesicle membrane. A PTDattached to another molecule facilitates the molecule traversing amembrane, for example going from extracellular space to intracellularspace, or cytosol to within an organelle. In some embodiments, a PTD iscovalently linked to the amino terminus of an IL-10 polypeptide, whilein other embodiments, a PTD is covalently linked to the carboxylterminus of an IL-10 polypeptide. Exemplary protein transduction domainsinclude, but are not limited to, a minimal undecapeptide proteintransduction domain (corresponding to residues 47-57 of HIV-1 TATcomprising YGRKKRRQRRR; SEQ ID NO:1); a polyarginine sequence comprisinga number of arginine residues sufficient to direct entry into a cell(e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); a VP22 domain(Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); a DrosophilaAntennapedia protein transduction domain (Noguchi et al. (2003) Diabetes52(7):1732-1737); a truncated human calcitonin peptide (Trehin et al.(2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. (2000)Proc. Natl. Acad. Sci. USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:2);Transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:3);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:4); and RQIKIWFQNRRMKWKK(SEQ ID NO:5). Exemplary PTDs include, but are not limited to,YGRKKRRQRRR (SEQ ID NO:1), RKKRRQRRR (SEQ ID NO:6); an argininehomopolymer of from 3 arginine residues to 50 arginine residues;exemplary PTD domain amino acid sequences include, but are not limitedto, any of the following: YGRKKRRQRRR (SEQ ID NO:1); RKKRRQRR (SEQ IDNO:7); YARAAARQARA (SEQ ID NO:8); THRLPRRRRRR (SEQ ID NO://9); andGGRRARRRRRR (SEQ ID NO:10).

The carboxyl group COR₃ of the amino acid at the C-terminal end of anIL-10 polypeptide can be present in a free form (R₃═OH) or in the formof a physiologically-tolerated alkaline or alkaline earth salt such as,e.g., a sodium, potassium or calcium salt. The carboxyl group can alsobe esterified with primary, secondary or tertiary alcohols such as,e.g., methanol, branched or unbranched C₁-C₆-alkyl alcohols, e.g., ethylalcohol or tert-butanol. The carboxyl group can also be amidated withprimary or secondary amines such as ammonia, branched or unbranchedC₁-C₆-alkylamines or C₁-C₆ di-alkylamines, e.g., methylamine ordimethylamine.

The amino group of the amino acid NR₁R₂ at the N-terminus of an IL-10polypeptide can be present in a free form (R₁═H and R₂═H) or in the formof a physiologically-tolerated salt such as, e.g., a chloride oracetate. The amino group can also be acetylated with acids such thatR₁═H and R₂═acetyl, trifluoroacetyl, or adamantyl. The amino group canbe present in a form protected by amino-protecting groups conventionallyused in peptide chemistry, such as those provided above (e.g., Fmoc,Benzyloxy-carbonyl (Z), Boc, and Alloc). The amino group can beN-alkylated in which R₁ and/or R₂═C₁-C₆ alkyl or C₂-C₈ alkenyl or C₇-C₉aralkyl. Alkyl residues can be straight-chained, branched or cyclic(e.g., ethyl, isopropyl and cyclohexyl, respectively).

Particular Modifications to Enhance and/or Mimic IL-10 Function

It is frequently beneficial, and sometimes imperative, to improve one ofmore physical properties of the treatment modalities disclosed herein(e.g., IL-10) and/or the manner in which they are administered.Improvements of physical properties include, for example, modulatingimmunogenicity; methods of increasing water solubility, bioavailability,serum half-life, and/or therapeutic half-life; and/or modulatingbiological activity. Certain modifications may also be useful to, forexample, raise of antibodies for use in detection assays (e.g., epitopetags) and to provide for ease of protein purification. Such improvementsmust generally be imparted without adversely impacting the bioactivityof the treatment modality and/or increasing its immunogenicity.

Pegylation of IL-10 is one particular modification contemplated by thepresent disclosure, while other modifications include, but are notlimited to, glycosylation (N- and O-linked); polysialylation; albuminfusion molecules comprising serum albumin (e.g., human serum albumin(HSA), cyno serum albumin, or bovine serum albumin (BSA)); albuminbinding through, for example a conjugated fatty acid chain (acylation);and Fc-fusion proteins.

Pegylation:

The clinical effectiveness of protein therapeutics is often limited byshort plasma half-life and susceptibility to protease degradation.Studies of various therapeutic proteins (e.g., filgrastim) have shownthat such difficulties may be overcome by various modifications,including conjugating or linking the polypeptide sequence to any of avariety of nonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes. This is frequently effectedby a linking moiety covalently bound to both the protein and thenonproteinaceous polymer, e.g., a PEG. Such PEG-conjugated biomoleculeshave been shown to possess clinically useful properties, includingbetter physical and thermal stability, protection against susceptibilityto enzymatic degradation, increased solubility, longer in vivocirculating half-life and decreased clearance, reduced immunogenicityand antigenicity, and reduced toxicity.

In addition to the beneficial effects of pegylation on pharmacokineticparameters, pegylation itself may enhance activity. For example,PEG-IL-10 has been shown to be more efficacious against certain cancersthan unpegylated IL-10 (see, e.g., EP 206636A2). Certain embodiments ofthe present disclosure contemplate the use of a relatively small PEG(e.g., 5 kDa) that improves the pharmacokinetic profile of the IL-10molecule without causing untoward adverse effects; such PEG-IL-10molecules are especially efficacious for chronic use.

PEGs suitable for conjugation to a polypeptide sequence are generallysoluble in water at room temperature, and have the general formulaR(O—CH₂—CH₂)_(n)O—R, where R is hydrogen or a protective group such asan alkyl or an alkanol group, and where n is an integer from 1 to 1000.When R is a protective group, it generally has from 1 to 8 carbons. ThePEG conjugated to the polypeptide sequence can be linear or branched.Branched PEG derivatives, “star-PEGs” and multi-armed PEGs arecontemplated by the present disclosure. A molecular weight of the PEGused in the present disclosure is not restricted to any particularrange, and examples are set forth elsewhere herein; by way of example,certain embodiments have molecular weights between 5 kDa and 20 kDa,while other embodiments have molecular weights between 4 kDa and 10 kDa.

The present disclosure also contemplates compositions of conjugateswherein the PEGs have different n values, and thus the various differentPEGs are present in specific ratios. For example, some compositionscomprise a mixture of conjugates where n=1, 2, 3 and 4. In somecompositions, the percentage of conjugates where n=1 is 18-25%, thepercentage of conjugates where n=2 is 50-66%, the percentage ofconjugates where n=3 is 12-16%, and the percentage of conjugates wheren=4 is up to 5%. Such compositions can be produced by reactionconditions and purification methods know in the art. Exemplary reactionconditions are described throughout the specification. Cation exchangechromatography may be used to separate conjugates, and a fraction isthen identified which contains the conjugate having, for example, thedesired number of PEGs attached, purified free from unmodified proteinsequences and from conjugates having other numbers of PEGs attached.

Pegylation most frequently occurs at the alpha amino group at theN-terminus of the polypeptide, the epsilon amino group on the side chainof lysine residues, and the imidazole group on the side chain ofhistidine residues. Since most recombinant polypeptides possess a singlealpha and a number of epsilon amino and imidazole groups, numerouspositional isomers can be generated depending on the linker chemistry.General pegylation strategies known in the art can be applied herein.PEG may be bound to a polypeptide of the present disclosure via aterminal reactive group (a “spacer”) which mediates a bond between thefree amino or carboxyl groups of one or more of the polypeptidesequences and polyethylene glycol. The PEG having the spacer which maybe bound to the free amino group includes N-hydroxysuccinylimidepolyethylene glycol which may be prepared by activating succinic acidester of polyethylene glycol with N-hydroxysuccinylimide. Anotheractivated polyethylene glycol which may be bound to a free amino groupis 2,4-bis(O-methoxypolyethyleneglycol)-6-chloro-s-triazine, which maybe prepared by reacting polyethylene glycol monomethyl ether withcyanuric chloride. The activated polyethylene glycol which is bound tothe free carboxyl group includes polyoxyethylenediamine.

Conjugation of one or more of the polypeptide sequences of the presentdisclosure to PEG having a spacer may be carried out by variousconventional methods. For example, the conjugation reaction can becarried out in solution at a pH of from 5 to 10, at temperature from 4°C. to room temperature, for 30 minutes to 20 hours, utilizing a molarratio of reagent to protein of from 4:1 to 30:1. Reaction conditions maybe selected to direct the reaction towards producing predominantly adesired degree of substitution. In general, low temperature, low pH(e.g., pH=5), and short reaction time tend to decrease the number ofPEGs attached, whereas high temperature, neutral to high pH (e.g.,pH≥7), and longer reaction time tend to increase the number of PEGsattached. Various means known in the art may be used to terminate thereaction. In some embodiments the reaction is terminated by acidifyingthe reaction mixture and freezing at, e.g., −20° C. Pegylation ofvarious molecules is discussed in, for example, U.S. Pat. Nos.5,252,714; 5,643,575; 5,919,455; 5,932,462; and 5,985,263. PEG-IL-10 isdescribed in, e.g., U.S. Pat. No. 7,052,686. Specific reactionconditions contemplated for use herein are set forth in the Experimentalsection.

The present disclosure also contemplates the use of PEG mimetics.Recombinant PEG mimetics have been developed that retain the attributesof PEG (e.g., enhanced serum half-life) while conferring severaladditional advantageous properties. By way of example, simplepolypeptide chains (comprising, for example, Ala, Glu, Gly, Pro, Ser andThr) capable of forming an extended conformation similar to PEG can beproduced recombinantly already fused to the peptide or protein drug ofinterest (e.g., Amunix' XTEN technology; Mountain View, Calif.). Thisobviates the need for an additional conjugation step during themanufacturing process. Moreover, established molecular biologytechniques enable control of the side chain composition of thepolypeptide chains, allowing optimization of immunogenicity andmanufacturing properties.

Glycosylation:

For purposes of the present disclosure, “glycosylation” is meant tobroadly refer to the enzymatic process that attaches glycans toproteins, lipids or other organic molecules. The use of the term“glycosylation” in conjunction with the present disclosure is generallyintended to mean adding or deleting one or more carbohydrate moieties(either by removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that may or may not be present in the nativesequence. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins involving a change in the natureand proportions of the various carbohydrate moieties present.

Glycosylation can dramatically affect the physical properties (e.g.,solubility) of polypeptides such as IL-10 and can also be important inprotein stability, secretion, and subcellular localization. Glycosylatedpolypeptides may also exhibit enhanced stability or may improve one ormore pharmacokinetic properties, such as half-life. In addition,solubility improvements can, for example, enable the generation offormulations more suitable for pharmaceutical administration thanformulations comprising the non-glycosylated polypeptide.

Addition of glycosylation sites can be accomplished by altering theamino acid sequence. The alteration to the polypeptide may be made, forexample, by the addition of, or substitution by, one or more serine orthreonine residues (for 0-linked glycosylation sites) or asparagineresidues (for N-linked glycosylation sites). The structures of N-linkedand O-linked oligosaccharides and the sugar residues found in each typemay be different. One type of sugar that is commonly found on both isN-acetylneuraminic acid (hereafter referred to as sialic acid). Sialicacid is usually the terminal residue of both N-linked and O-linkedoligosaccharides and, by virtue of its negative charge, may conferacidic properties to the glycoprotein. A particular embodiment of thepresent disclosure comprises the generation and use of N-glycosylationvariants.

The polypeptide sequences of the present disclosure may optionally bealtered through changes at the nucleic acid level, particularly bymutating the nucleic acid encoding the polypeptide at preselected basessuch that codons are generated that will translate into the desiredamino acids.

Polysialylation:

The present disclosure also contemplates the use of polysialylation, theconjugation of polypeptides to the naturally occurring, biodegradableα-(2→8) linked polysialic acid (“PSA”) in order to improve thepolypeptides' stability and in vivo pharmacokinetics.

Albumin Fusion:

Additional suitable components and molecules for conjugation includealbumins such as human serum albumin (HSA), cyno serum albumin, andbovine serum albumin (BSA).

According to the present disclosure, albumin may be conjugated to a drugmolecule (e.g., a polypeptide described herein) at the carboxylterminus, the amino terminus, both the carboxyl and amino termini, andinternally (see, e.g., U.S. Pat. Nos. 5,876,969 and 7,056,701).

In the HSA-drug molecule conjugates contemplated by the presentdisclosure, various forms of albumin may be used, such as albuminsecretion pre-sequences and variants thereof, fragments and variantsthereof, and HSA variants. Such forms generally possess one or moredesired albumin activities. In additional embodiments, the presentdisclosure involves fusion proteins comprising a polypeptide drugmolecule fused directly or indirectly to albumin, an albumin fragment,and albumin variant, etc., wherein the fusion protein has a higherplasma stability than the unfused drug molecule and/or the fusionprotein retains the therapeutic activity of the unfused drug molecule.In some embodiments, the indirect fusion is effected by a linker, suchas a peptide linker or modified version thereof.

As alluded to above, fusion of albumin to one or more polypeptides ofthe present disclosure can, for example, be achieved by geneticmanipulation, such that the nucleic acid coding for HSA, or a fragmentthereof, is joined to the nucleic acid coding for the one or morepolypeptide sequences.

Alternative Albumin Binding Strategies:

Several albumin-binding strategies have been developed as alternativesto direct fusion and may be used with the IL-10 agents described herein.By way of example, the present disclosure contemplates albumin bindingthrough a conjugated fatty acid chain (acylation) and fusion proteinswhich comprise an albumin binding domain (ABD) polypeptide sequence andthe sequence of one or more of the polypeptides described herein.

Conjugation with Other Molecules:

Additional suitable components and molecules for conjugation include,for example, thyroglobulin; tetanus toxoid; Diphtheria toxoid; polyaminoacids such as poly(D-lysine:D-glutamic acid); VP6 polypeptides ofrotaviruses; influenza virus hemaglutinin, influenza virusnucleoprotein; Keyhole Limpet Hemocyanin (KLH); and hepatitis B viruscore protein and surface antigen; or any combination of the foregoing.

Thus, the present disclosure contemplates conjugation of one or moreadditional components or molecules at the N- and/or C-terminus of apolypeptide sequence, such as another polypeptide (e.g., a polypeptidehaving an amino acid sequence heterologous to the subject polypeptide),or a carrier molecule. Thus, an exemplary polypeptide sequence can beprovided as a conjugate with another component or molecule.

An IL-10 polypeptide may also be conjugated to large, slowly metabolizedmacromolecules such as proteins; polysaccharides, such as sepharose,agarose, cellulose, or cellulose beads; polymeric amino acids such aspolyglutamic acid, or polylysine; amino acid copolymers; inactivatedvirus particles; inactivated bacterial toxins such as toxoid fromdiphtheria, tetanus, cholera, or leukotoxin molecules; inactivatedbacteria; and dendritic cells. Such conjugated forms, if desired, can beused to produce antibodies against a polypeptide of the presentdisclosure.

Additional candidate components and molecules for conjugation includethose suitable for isolation or purification. Particular non-limitingexamples include binding molecules, such as biotin (biotin-avidinspecific binding pair), an antibody, a receptor, a ligand, a lectin, ormolecules that comprise a solid support, including, for example, plasticor polystyrene beads, plates or beads, magnetic beads, test strips, andmembranes.

Fc-Fusion Molecules:

In certain embodiments, the amino- or carboxyl-terminus of a polypeptidesequence of the present disclosure can be fused with an immunoglobulinFc region (e.g., human Fc) to form a fusion conjugate (or fusionmolecule). Fc fusion conjugates have been shown to increase the systemichalf-life of biopharmaceuticals, and thus the biopharmaceutical productmay require less frequent administration.

Fc binds to the neonatal Fc receptor (FcRn) in endothelial cells thatline the blood vessels, and, upon binding, the Fc fusion molecule isprotected from degradation and re-released into the circulation, keepingthe molecule in circulation longer. This Fc binding is believed to bethe mechanism by which endogenous IgG retains its long plasma half-life.More recent Fc-fusion technology links a single copy of abiopharmaceutical to the Fc region of an antibody to optimize thepharmacokinetic and pharmacodynamic properties of the biopharmaceuticalas compared to traditional Fc-fusion conjugates.

Other Modifications:

The present disclosure contemplates the use of other modifications,currently known or developed in the future, of IL-10 to improve one ormore properties. One such method involves modification of thepolypeptide sequences by hesylation, which utilizes hydroxyethyl starchderivatives linked to other molecules in order to modify the polypeptidesequences' characteristics. Various aspects of hesylation are describedin, for example, U.S. Patent Appln. Nos. 2007/0134197 and 2006/0258607.

The present disclosure also contemplates fusion molecules comprisingSmall Ubiquitin-like Modifier (SUMO) as a fusion tag (LifeSensors, Inc.;Malvern, Pa.). Fusion of a polypeptide described herein to SUMO mayconvey several beneficial effects, including enhancement of expression,improvement in solubility, and/or assistance in the development ofpurification methods. SUMO proteases recognize the tertiary structure ofSUMO and cleave the fusion protein at the C-terminus of SUMO, thusreleasing a polypeptide described herein with the desired N-terminalamino acid.

The present disclosure also contemplates the use of PASylation™(XL-Protein GmbH (Freising, Germany)). This technology expands theapparent molecular size of a protein of interest, without having anegative impact on the therapeutic bioactivity of the protein, beyondthe pore size of the renal glomeruli, thereby decreasing renal clearanceof the protein.

Linkers:

Linkers and their use have been described above. Any of the foregoingcomponents and molecules used to modify the polypeptide sequences of thepresent disclosure may optionally be conjugated via a linker. Suitablelinkers include “flexible linkers” which are generally of sufficientlength to permit some movement between the modified polypeptidesequences and the linked components and molecules. The linker moleculesare generally about 6-50 atoms long. The linker molecules may also be,for example, aryl acetylene, ethylene glycol oligomers containing 2-10monomer units, diamines, diacids, amino acids, or combinations thereof.Suitable linkers can be readily selected and can be of any suitablelength, such as 1 amino acid (e.g., Gly), 2, 3, 4, 5, 6, 7, 8, 9, 10,10-20, 20-30, 30-50 or more than 50 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (for example, (GS)_(n), GSGGS_(n), GGGS_(n),(G_(m)S_(o))_(n), (G_(m)S_(o)G_(m))_(n),(G_(m)S_(o)G_(m)S_(o)G_(m))_(n), (GSGGS_(m))_(n), (GSGS_(m)G)_(n) and(GGGS_(m))_(n), and combinations thereof, where m, and and o are eachindependently selected from an integer of at least one), glycine-alaninepolymers, alanine-serine polymers, and other flexible linkers. Glycineand glycine-serine polymers are relatively unstructured, and thereforemay serve as a neutral tether between components. Exemplary flexiblelinkers include, but are not limited to GGSG, GGSGG, GSGSG, GSGGG,GGGSG, and GSSSG.

Therapeutic and Prophylactic Uses

The present disclosure contemplates the use of the IL-10 polypeptidesdescribed herein (e.g., PEG-IL-10) in the treatment and/or prevention ofdiseases, disorders or conditions, and/or the symptoms thereof, relatingto, or resulting from, for example, hypercholesterolemia, aberrant lipidprofile, and other disorders associated, directly or indirectly, withcholesterol homeostasis. While particular uses are described in detailhereafter, it is to be understood that the present disclosure is not solimited. In addition, although specific categories of exemplarydiseases, disorders and conditions associated with, or resulting from,hypercholesterolemia and aberrant lipid profile are discussed hereafter,it is to be understood that there is often overlap between one or morecategories (e.g., certain cardiovascular diseases may have aninflammatory component).

Cardiovascular Diseases.

In particular embodiments, the present disclosure contemplates the useof the IL-10 polypeptides (e.g., PEG-IL-10) described herein to treatand/or prevent cardiovascular diseases, disorders and conditions, aswell as disorders associated therewith, resulting fromhypercholesterolemia and aberrant lipid profile.

As used herein, the terms “cardiovascular disease”, “heart disease” andthe like refer to any disease that affects the cardiovascular system,primarily cardiac disease, vascular diseases of the brain and kidney,and peripheral arterial diseases. Cardiovascular disease is aconstellation of diseases that includes coronary heart disease (e.g.,ischemic heart disease or coronary artery disease), atherosclerosis,cardiomyopathy, hypertension, hypertensive heart disease, cor pulmonale,cardiac dysrhythmias, endocarditis, cerebrovascular disease, andperipheral arterial disease. Cardiovascular disease is the leading causeof deaths worldwide, and while it usually affects older adults, theantecedents of cardiovascular disease, notably atherosclerosis, begin inearly life.

Particular embodiments of the present disclosure are directed to the useof the IL-10 polypeptides described herein to treat and/or preventatherosclerosis, a chronic condition in which an arterial wall thickensdue to formation of plaques as a result of the accumulation of fattymaterials such as cholesterol and triglycerides. As discussed furtherherein, atherosclerosis frequently involves a chronic inflammatoryresponse in the walls of arteries, caused largely by the accumulation ofmacrophages and promoted by LDLs without adequate removal of fats andcholesterol from the macrophages by functional HDLs. Chronicallyexpanding atherosclerotic lesions can cause complete closure of thelumen, which may only manifest when the lumen stenosis is so severe thatblood supply to downstream tissue(s) is insufficient, resulting inischemia.

Particularly contemplated by the present disclosure are embodimentswherein the cardiovascular disease comprises a hyperlipidemia (orhyperlipoproteinemia), conditions characterized by abnormally elevatedlevels of lipids and/or lipoproteins in the blood. Hyperlipidemias maybe classified as familial (or primary) when caused by specific geneticabnormalities, acquired (or secondary) when resulting from anotherunderlying disorder, or idiopathic, when of unknown cause.Hyperlipidemias may also be classified based on which types of lipidsand/or lipoproteins are elevated. Non-limiting examples ofhyperlipidemias include dyslipidemia, hypercholesterolemia,hyperglyceridemia, hypertriglyceridemia, hyperlipoproteinemia,hyperchylomicronemia, and combined hyperlipidemia. Hyperlipoproteinemiasinclude, for example, hyperlipoproteinemia type Ia, hyperlipoproteinemiatype Ib, hyperlipoproteinemia type Ic, hyperlipoproteinemia type IIa,hyperlipoproteinemia type IIb, hyperlipoproteinemia type III,hyperlipoproteinemia type IV, and hyperlipoproteinemia type V.

In particular embodiments, the present disclosure contemplates thetreatment and/or prevention of familial hypercholesterolemia (FH), agenetic disorder characterized by very high levels of LDL in the blood.FH is associated with early cardiovascular disease, as accelerateddeposition of cholesterol in the arterial walls leads toatherosclerosis. In certain patient populations, total cholesterollevels range from 350-550 mg/dL, while in other patient populations theyrange from of 650-1000 mg/dL. In obese patients having FH, cholesterollevels can be dramatically higher.

Attempts to treat cardiovascular disease by controlling levels of lipidsand/or lipoproteins in the blood have met with limited success. Forexample, although administration of statins reduces cardiovascular riskin some individuals, these therapeutic compounds do not reducetriglyceride levels. In individuals at cardiovascular risk who exhibitdeleteriously high levels of triglycerides, a member of the fibrateclass of therapeutic agents may be administered. However, althoughlowering triglyceride and LDL levels, fibrates do not affect HDL levels.Moreover, combination treatments involving statins and fibrates, whilesometimes effective, often cause a significant increase in the risk ofmyopathy and rhabdomyolysis, and therefore can only be carried out undervery close medical supervision. In view of limitations as exemplifiedabove, there is clearly a need for improved agents for the use andtreatment of cardiovascular diseases, including those associated withhigh lipid and/or lipoprotein levels.

Thrombosis and Thrombotic Conditions.

In other embodiments, the present disclosure contemplates the use of theIL-10 polypeptides (e.g., PEG-IL-10) described herein to treat and/orprevent thrombosis and thrombotic diseases, disorders and conditions, aswell as disorders associated therewith, resulting fromhypercholesterolemia and aberrant lipid profile. Thrombosis, theformation of a thrombus inside a blood vessel resulting in obstructionof the flow of blood through the circulatory system, may be caused byabnormalities in one or more of the following (Virchow's triad):hypercoagulability or increased blood clotting, endothelial cell injury,or disturbed blood flow (stasis, turbulence).

Thrombosis is generally categorized as venous or arterial, each of whichcan be presented by several subtypes. Venous thrombosis includes deepvein thrombosis (DVT), portal vein thrombosis, renal vein thrombosis,jugular vein thrombosis, Budd-Chiari syndrome, Paget-Schroetter disease,and cerebral venous sinus thrombosis. Arterial thrombosis includesstroke and myocardial infarction.

Inflammatory Disorders.

When cholesterol and/or LDL become embedded in the walls of bloodvessels, an immune response can be triggered, which, in turn, results inchronic inflammation. In response to this inflammation, blood monocytesadhere to the endothelium, transmigrate into the subendothelial space,and differentiate toward macrophages. Macrophages, in turn, engulf thecholesterol deposits and modified LDL by phagocytosis via scavengerreceptors, which are distinct from LDL receptors. However, the adaptivemechanisms mediated by macrophages are not sufficient to process theuncontrolled cholesterol and/or LDL deposition seen under pathologicconditions. As a result, the lipid-laden macrophages transform into“foam cells”, often accompanied by release of inflammation-inducingmolecules. Both cholesterol/LDL deposition and the attendant foamcell-mediated pro-inflammatory reactions in the walls of the bloodvessels lead to the development of atherosclerotic lesions. Thus, oneconsequence of modulating the levels of a lipid or lipoprotein is thereduction or elimination of a chronic inflammation.

The present disclosure includes embodiments wherein the IL-10 agentsdescribed herein (e.g., PEG-IL-10) are used in the treatment and/orprevention of a vasculitis. Vasculitis is a varied group of disordersfeaturing inflammation of a vessel wall including lymphatic vessels andblood vessels like veins (phlebitis), arteries (arteritis) andcapillaries due to leukocyte migration and resultant damage. Theinflammation may affect arteries and/or veins, regardless of size. Itmay be focal or widespread, with areas of inflammation scatteredthroughout a particular organ or tissue, or even affecting more than oneorgan system in the body. Vasculitis includes, without limitation,Buerger's disease (thromboangiitis obliterans), cerebral vasculitis(central nervous system vasculitis), Churg-Strauss arteritis,cryoglobulinemia, essential cryoglobulinemic vasculitis, giant cell(temporal) arteritis, Henoch-Schonlein purpura, hypersensitivityvasculitis (allergic vasculitis), Kawasaki disease, microscopicpolyarteritis/polyangiitis, polyarteritis nodosa, polymyalgia rheumatica(PMR), rheumatoid vasculitis, Takayasu arteritis, thrombophlebitis,Wegener's granulomatosis; and vasculitis secondary to connective tissuedisorders like systemic lupus erythematosus, rheumatoid arthritis,relapsing polychondritis, Behcet's disease, or other connective tissuedisorders; and vasculitis secondary to viral infection.

Other embodiments are directed to an inflammatory heart disease, whichrefers to a condition characterized by inflammation of the heart muscleand/or the surrounding tissue. Examples include, but are not limited to,endocarditis, inflammatory cardiomegaly, and myocarditis.

Fibrotic Disorders:

The present disclosure also provides methods of treating or preventingfibrotic diseases, disorders and conditions. As used herein, the phrase“fibrotic diseases, disorders and conditions”, and similar terms (e.g.,“fibrotic disorders”) and phrases, is to be construed broadly such thatit includes any condition which may result in the formation of fibrotictissue or scar tissue (e.g., fibrosis in one or more tissues). By way ofexample, injuries (e.g., wounds) that may give rise to scar tissueinclude wounds to the skin, eye, lung, kidney, liver, central nervoussystem, and cardiovascular system. The phrase also encompasses scartissue formation resulting from stroke, and tissue adhesion, forexample, as a result of injury or surgery.

As used herein the term “fibrosis” refers to the formation of fibroustissue as a reparative or reactive process, rather than as a normalconstituent of an organ or tissue. Fibrosis is characterized byfibroblast accumulation and collagen deposition in excess of normaldeposition in any particular tissue.

Fibrotic disorders include, but are not limited to, fibrosis arisingfrom wound healing, systemic and local scleroderma, atherosclerosis,restenosis, pulmonary inflammation and fibrosis, idiopathic pulmonaryfibrosis, interstitial lung disease, liver cirrhosis, fibrosis as aresult of chronic hepatitis B or C infection, kidney disease (e.g.,glomerulonephritis), heart disease resulting from scar tissue, keloidsand hypertrophic scars, and eye diseases such as macular degeneration,and retinal and vitreal retinopathy. Additional fibrotic diseasesinclude chemotherapeutic drug-induced fibrosis, radiation-inducedfibrosis, and injuries and burns.

Fibrotic disorders are often hepatic-related, and there is frequently anexus between such disorders and the inappropriate accumulation of livercholesterol and triglycerides within the hepatocytes and Kupffer cells.This accumulation appears to result in a pro-inflammatory response thatleads to liver fibrosis and cirrhosis. Hepatic disorders having afibrotic component include non-alcoholic fatty liver disease (NAFLD) andnon-alcoholic steatohepatitis (NASH). NAFLD occurs when steatosis (fatdeposition in the liver) is present that is not due to excessive alcoholuse. It is related to insulin resistance and the metabolic syndrome.NASH is the most extreme form of NAFLD, and is regarded as a major causeof cirrhosis of the liver of unknown cause.

Pharmaceutical Compositions

The IL-10 polypeptides of the present disclosure may be in the form ofcompositions suitable for administration to a subject. In general, suchcompositions are “pharmaceutical compositions” comprising IL-10 and oneor more pharmaceutically acceptable or physiologically acceptablediluents, carriers or excipients. In certain embodiments, the IL-10polypeptides are present in a therapeutically acceptable amount. Thepharmaceutical compositions may be used in the methods of the presentdisclosure; thus, for example, the pharmaceutical compositions can beadministered ex vivo or in vivo to a subject in order to practice thetherapeutic and prophylactic methods and uses described herein.

The pharmaceutical compositions of the present disclosure can beformulated to be compatible with the intended method or route ofadministration; exemplary routes of administration are set forth herein.Furthermore, the pharmaceutical compositions may be used in combinationwith other therapeutically active agents or compounds as describedherein in order to treat or prevent the diseases, disorders andconditions as contemplated by the present disclosure.

The pharmaceutical compositions typically comprise a therapeuticallyeffective amount of an IL-10 polypeptide contemplated by the presentdisclosure and one or more pharmaceutically and physiologicallyacceptable formulation agents. Suitable pharmaceutically acceptable orphysiologically acceptable diluents, carriers or excipients include, butare not limited to, antioxidants (e.g., ascorbic acid and sodiumbisulfate), preservatives (e.g., benzyl alcohol, methyl parabens, ethylor n-propyl, p-hydroxybenzoate), emulsifying agents, suspending agents,dispersing agents, solvents, fillers, bulking agents, detergents,buffers, vehicles, diluents, and/or adjuvants. For example, a suitablevehicle may be physiological saline solution or citrate buffered saline,possibly supplemented with other materials common in pharmaceuticalcompositions for parenteral administration. Neutral buffered saline orsaline mixed with serum albumin are further exemplary vehicles. Thoseskilled in the art will readily recognize a variety of buffers that canbe used in the pharmaceutical compositions and dosage forms contemplatedherein. Typical buffers include, but are not limited to,pharmaceutically acceptable weak acids, weak bases, or mixtures thereof.As an example, the buffer components can be water soluble materials suchas phosphoric acid, tartaric acids, lactic acid, succinic acid, citricacid, acetic acid, ascorbic acid, aspartic acid, glutamic acid, andsalts thereof. Acceptable buffering agents include, for example, a Trisbuffer, N-(2-Hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid) (HEPES),2-(N-Morpholino)ethanesulfonic acid (MES),2-(N-Morpholino)ethanesulfonic acid sodium salt (MES),3-(N-Morpholino)propanesulfonic acid (MOPS), andN-tris[Hydroxymethyl]methyl-3-aminopropanesulfonic acid (TAPS).

After a pharmaceutical composition has been formulated, it may be storedin sterile vials as a solution, suspension, gel, emulsion, solid, ordehydrated or lyophilized powder. Such formulations may be stored eitherin a ready-to-use form, a lyophilized form requiring reconstitutionprior to use, a liquid form requiring dilution prior to use, or otheracceptable form. In some embodiments, the pharmaceutical composition isprovided in a single-use container (e.g., a single-use vial, ampoule,syringe, or autoinjector (similar to, e.g., an EpiPen®)), whereas amulti-use container (e.g., a multi-use vial) is provided in otherembodiments. Any drug delivery apparatus may be used to deliver IL-10,including implants (e.g., implantable pumps) and catheter systems, slowinjection pumps and devices, all of which are well known to the skilledartisan. Depot injections, which are generally administeredsubcutaneously or intramuscularly, may also be utilized to release thepolypeptides disclosed herein over a defined period of time. Depotinjections are usually either solid- or oil-based and generally compriseat least one of the formulation components set forth herein. One ofordinary skill in the art is familiar with possible formulations anduses of depot injections.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleagenous suspension. This suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents mentioned herein. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butane diol. Acceptable diluents,solvents and dispersion media that may be employed include water,Ringer's solution, isotonic sodium chloride solution, Cremophor EL™(BASF, Parsippany, N.J.) or phosphate buffered saline (PBS), ethanol,polyol (e.g., glycerol, propylene glycol, and liquid polyethyleneglycol), and suitable mixtures thereof. In addition, sterile, fixed oilsare conventionally employed as a solvent or suspending medium. For thispurpose any bland fixed oil may be employed, including synthetic mono-or diglycerides. Moreover, fatty acids such as oleic acid, find use inthe preparation of injectables. Prolonged absorption of particularinjectable formulations can be achieved by including an agent thatdelays absorption (e.g., aluminum monostearate or gelatin).

The pharmaceutical compositions containing the active ingredient may bein a form suitable for oral use, for example, as tablets, capsules,troches, lozenges, aqueous or oily suspensions, dispersible powders orgranules, emulsions, hard or soft capsules, or syrups, solutions,microbeads or elixirs. In particular embodiments, an active ingredientof an agent co-administered with an IL-10 agent described herein is in aform suitable for oral use. Pharmaceutical compositions intended fororal use may be prepared according to any method known to the art forthe manufacture of pharmaceutical compositions, and such compositionsmay contain one or more agents such as, for example, sweetening agents,flavoring agents, coloring agents and preserving agents in order toprovide pharmaceutically elegant and palatable preparations. Tablets,capsules and the like contain the active ingredient in admixture withnon-toxic pharmaceutically acceptable excipients which are suitable forthe manufacture of tablets. These excipients may be, for example,diluents, such as calcium carbonate, sodium carbonate, lactose, calciumphosphate or sodium phosphate; granulating and disintegrating agents,for example, corn starch, or alginic acid; binding agents, for examplestarch, gelatin or acacia, and lubricating agents, for example magnesiumstearate, stearic acid or talc.

The tablets, capsules and the like suitable for oral administration maybe uncoated or coated by known techniques to delay disintegration andabsorption in the gastrointestinal tract and thereby provide a sustainedaction. For example, a time-delay material such as glyceryl monostearateor glyceryl distearate may be employed. They may also be coated bytechniques known in the art to form osmotic therapeutic tablets forcontrolled release. Additional agents include biodegradable orbiocompatible particles or a polymeric substance such as polyesters,polyamine acids, hydrogel, polyvinyl pyrrolidone, polyanhydrides,polyglycolic acid, ethylenevinylacetate, methylcellulose,carboxymethylcellulose, protamine sulfate, or lactide/glycolidecopolymers, polylactide/glycolide copolymers, or ethylenevinylacetatecopolymers in order to control delivery of an administered composition.For example, the oral agent can be entrapped in microcapsules preparedby coacervation techniques or by interfacial polymerization, by the useof hydroxymethylcellulose or gelatin-microcapsules or poly(methylmethacrolate) microcapsules, respectively, or in a colloid drugdelivery system. Colloidal dispersion systems include macromoleculecomplexes, nano-capsules, microspheres, microbeads, and lipid-basedsystems, including oil-in-water emulsions, micelles, mixed micelles, andliposomes. Methods for the preparation of the above-mentionedformulations will be apparent to those skilled in the art.

Formulations for oral use may also be presented as hard gelatin capsuleswherein the active ingredient is mixed with an inert solid diluent, forexample, calcium carbonate, calcium phosphate, kaolin ormicrocrystalline cellulose, or as soft gelatin capsules wherein theactive ingredient is mixed with water or an oil medium, for examplepeanut oil, liquid paraffin, or olive oil.

Aqueous suspensions contain the active materials in admixture withexcipients suitable for the manufacture thereof. Such excipients can besuspending agents, for example sodium carboxymethylcellulose,methylcellulose, hydroxy-propylmethylcellulose, sodium alginate,polyvinyl-pyrrolidone, gum tragacanth and gum acacia; dispersing orwetting agents, for example a naturally-occurring phosphatide (e.g.,lecithin), or condensation products of an alkylene oxide with fattyacids (e.g., polyoxy-ethylene stearate), or condensation products ofethylene oxide with long chain aliphatic alcohols (e.g., forheptadecaethyleneoxycetanol), or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol (e.g.,polyoxyethylene sorbitol monooleate), or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides (e.g., polyethylene sorbitan monooleate). The aqueoussuspensions may also contain one or more preservatives.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavoring agents may be added to provide a palatable oralpreparation.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active ingredient inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified herein.

The pharmaceutical compositions of the present disclosure may also be inthe form of oil-in-water emulsions. The oily phase may be a vegetableoil, for example olive oil or arachis oil, or a mineral oil, forexample, liquid paraffin, or mixtures of these. Suitable emulsifyingagents may be naturally occurring gums, for example, gum acacia or gumtragacanth; naturally occurring phosphatides, for example, soy bean,lecithin, and esters or partial esters derived from fatty acids; hexitolanhydrides, for example, sorbitan monooleate; and condensation productsof partial esters with ethylene oxide, for example, polyoxyethylenesorbitan monooleate.

Formulations can also include carriers to protect the compositionagainst rapid degradation or elimination from the body, such as acontrolled release formulation, including implants, liposomes,hydrogels, prodrugs and microencapsulated delivery systems. For example,a time delay material such as glyceryl monostearate or glyceryl stearatealone, or in combination with a wax, may be employed.

The present disclosure contemplates the administration of the IL-10polypeptides in the form of suppositories for rectal administration. Thesuppositories can be prepared by mixing the drug with a suitablenon-irritating excipient which is solid at ordinary temperatures butliquid at the rectal temperature and will therefore melt in the rectumto release the drug. Such materials include, but are not limited to,cocoa butter and polyethylene glycols.

The IL-10 polypeptides contemplated by the present disclosure may be inthe form of any other suitable pharmaceutical composition (e.g., spraysfor nasal or inhalation use) currently known or developed in the future.

The concentration of a polypeptide or fragment thereof in a formulationcan vary widely (e.g., from less than about 0.1%, usually at or at leastabout 2% to as much as 20% to 50% or more by weight) and will usually beselected primarily based on fluid volumes, viscosities, andsubject-based factors in accordance with, for example, the particularmode of administration selected.

Routes of Administration

The present disclosure contemplates the administration of IL-10 (e.g.,IL-10 polypeptide), and compositions thereof, in any appropriate manner.Suitable routes of administration include parenteral (e.g.,intramuscular, intravenous, subcutaneous (e.g., injection or implant),intraperitoneal, intracisternal, intraarticular, intraperitoneal,intracerebral (intraparenchymal) and intracerebroventricular), oral,nasal, vaginal, sublingual, intraocular, rectal, topical (e.g.,transdermal), sublingual and inhalation. Depot injections, which aregenerally administered subcutaneously or intramuscularly, may also beutilized to release the IL-10 polypeptides disclosed herein over adefined period of time.

Particular embodiments of the present disclosure contemplate parenteraladministration. In some particular embodiments, the parenteraladministration is intravenous, and in other particular embodiments theparenteral administration is subcutaneous.

Combination Therapy

The present disclosure contemplates the use of IL-10 (e.g., PEG-IL-10)in combination with one or more active therapeutic agents or otherprophylactic or therapeutic modalities (e.g., radiation). In suchcombination therapy, the various active agents frequently have differentmechanisms of action than IL-10. Such combination therapy may beespecially advantageous by allowing a dose reduction of one or more ofthe agents, thereby reducing or eliminating the adverse effectsassociated with one or more of the agents; furthermore, such combinationtherapy may have a synergistic therapeutic or prophylactic effect on theunderlying disease, disorder, or condition.

In particular embodiments, the present disclosure provides methods fortreating and/or preventing diseases, disorders or conditions associatedwith (either directly or indirectly) cholesterol homeostasis, includingassociated cardiovascular, thrombotic and inflammatory disorders, withthe IL-10 polypeptides described herein (e.g., PEG-IL-10) and at leastone additional therapeutic or diagnostic agent. It is to be understoodthat combination therapy is not limited to agents that treat and/orprevent the aforementioned diseases, disorders and conditions; forexample, agents contemplated for use in combination with the IL-10polypeptides may have efficacy in treating or preventing other metabolicdisorders, such as diabetes or obesity. Use of the IL-10 polypeptides(e.g., PEG-IL-10) in combination with modified diets and/or exerciseregimens is also contemplated herein.

As used herein, “combination” is meant to include therapies that can beadministered separately, for example, formulated separately for separateadministration (e.g., as may be provided in a kit), and therapies thatcan be administered together in a single formulation (i.e., a“co-formulation”).

In certain embodiments, the IL-10 polypeptides are administered orapplied sequentially, e.g., where one agent is administered prior to oneor more other agents. In other embodiments, the IL-10 polypeptides areadministered simultaneously, e.g., where two or more agents areadministered at or about the same time; the two or more agents may bepresent in two or more separate formulations or combined into a singleformulation (i.e., a co-formulation). Regardless of whether the two ormore agents are administered sequentially or simultaneously, they areconsidered to be administered in combination for purposes of the presentdisclosure.

The IL-10 polypeptides of the present disclosure may be used incombination with at least one active agent in any manner appropriateunder the circumstances. In one embodiment, treatment with the at leastone active agent and at least one IL-10 polypeptide (i.e., homodimer) ofthe present disclosure is maintained over a period of time. In anotherembodiment, treatment with the at least one active agent is reduced ordiscontinued (e.g., when the subject is stable), while treatment withthe IL-10 polypeptide of the present disclosure is maintained at aconstant dosing regimen. In a further embodiment, treatment with the atleast one active agent is reduced or discontinued (e.g., when thesubject is stable), while treatment with the IL-10 polypeptide of thepresent disclosure is reduced (e.g., lower dose, less frequent dosing orshorter treatment regimen). In yet another embodiment, treatment withthe at least one active agent is reduced or discontinued (e.g., when thesubject is stable), and treatment with the IL-10 polypeptide of thepresent disclosure is increased (e.g., higher dose, more frequent dosingor longer treatment regimen). In yet another embodiment, treatment withthe at least one active agent is maintained and treatment with the IL-10polypeptide of the present disclosure is discontinued or reduced (e.g.,lower dose, less frequent dosing or shorter treatment regimen). In yetanother embodiment, treatment with the at least one active agent andtreatment with the IL-10 polypeptide of the present disclosure arediscontinued or reduced (e.g., lower dose, less frequent dosing orshorter treatment regimen).

While particular agents suitable for use in combination with the IL-10polypeptides (e.g., PEG-IL-10) disclosed herein are set forth hereafter,it is to be understood that the present disclosure is not so limited.Hereafter, certain agents are set forth in specific categories ofexemplary diseases, disorders and conditions; however, it is to beunderstood that there is often overlap between one or more categories(e.g., certain agents may have both cardiovascular and anti-inflammatoryeffects).

Cholesterol Homeostasis Agents.

Particular embodiments of the present disclosure involve combinations ofIL-10 polypeptides with agents associated with cholesterol homeostasis.Many of these agents target different pathways involving the absorption,synthesis, transport, storage, catabolism, and excretion of cholesterol,and are thus particularly useful candidates for combination therapy.

Examples of therapeutic agents useful in combination therapy for thetreatment of hypercholesterolemia (and thus frequently atherosclerosis,for example) include statins (e.g., CRESTOR, LESCOL, LIPITOR, MEVACOR,PRAVACOL, and ZOCOR), which inhibit the enzymatic synthesis ofcholesterol; bile acid resins (e.g., COLESTID, LO-CHOLEST, PREVALITE,QUESTRAN, and WELCHOL), which sequester cholesterol and prevent itsabsorption; ezetimibe (ZETIA), which blocks cholesterol absorption;fibric acid (e.g., TRICOR), which reduce triglycerides and may modestlyincrease HDL; niacin (e.g., NIACOR), which modestly lowers LDLcholesterol and triglycerides; and/or a combination of theaforementioned (e.g., VYTORIN (ezetimibe with simvastatin). Alternativecholesterol treatments that may be candidates for use in combinationwith the IL-10 polypeptides described herein include various supplementsand herbs (e.g., garlic, policosanol, and guggul). Several classes ofthe aforementioned therapeutic agents are discussed further hereafter.

Particular embodiments of the present disclosure comprise an IL-10 agentin combination with a fibrate. Fibrates, a class of amphipathiccarboxylic acids, may be used as anti-hyperlipidemic agents to decreaselevels of, e.g., triglycerides and LDL, and to increase levels of HDL.Examples of suitable fibrates include, without limitation, Bezafibrate,Ciprofibrate, Clofibrate, Gemfibrozil, and Fenofibrate.

Further particular embodiments of the present disclosure comprise anIL-10 agent in combination with a HMG-CoA Reductase Inhibitor (astatin). HMG-CoA Reductase Inhibitors may lower LDL and/or cholesterollevels by inhibiting the enzyme HMG-CoA Reductase, which plays a centralrole in the production of cholesterol in the liver. To compensate forthe decreased cholesterol availability, synthesis of hepatic LDLreceptors is increased, resulting in increased clearance of LDLparticles from the blood. Examples of suitable statins include, withoutlimitation, Atorvastatin, Fluvastatin, Lovastatin, Pitavastatin,Pravastatin, Rosuvastatin, and Simvastatin. Combinations of IL-10polypeptides with a statin are particularly contemplated herein.

Still further particular embodiments of the present disclosure comprisean IL-10 agent in combination with a niacin. Niacins may lower LDLlevels by selectively inhibiting hepatic diacyglycerolacyltransferase-2; reducing triglyceride synthesis, and reducing VLDLsecretion through a receptor HM74 and HM74A or GPR109A. A non-limitinguse of a niacin is as an anti-hyperlipidemic agent to inhibit thebreakdown of fats in adipose tissue. By blocking the breakdown of fats,a niacin causes a decrease in free fatty acids in the blood and, as aconsequence, decreases the secretion of VLDL and cholesterol by theliver. By lowering VLDL levels, a niacin may also increase the level ofHDL in blood. Examples of suitable niacins include, without limitation,acipimox, niacin, nicotinamide, and vitamin B3.

Other particular embodiments of the present disclosure comprise an IL-10agent in combination with a bile acid sequestrant. Bile acidsequestrants (also known as resins) bind certain components of bile inthe gastrointestinal tract, thereby disrupting the enterohepaticcirculation of bile acids by sequestering them and preventing theirreabsorption from the gut. Bile acid sequestrants are particularlyeffective for lowering LDL and cholesterol, and may also raise HDLlevels. Examples of suitable bile acid sequestrants include, withoutlimitation, Cholestyramine, Colesevelam, and Colestipol.

Additional particular embodiments of the present disclosure comprise anIL-10 agent in combination with a cholesterol absorption inhibitor.Cholesterol absorption inhibitors decrease absorption of cholesterolfrom the intestine; this leads to up-regulation of LDL-receptors on thesurface of cells and increased LDL cholesterol uptake into these cells,thus decreasing levels of LDL in the blood plasma. Examples of suitablecholesterol absorption inhibitors include, without limitation,ezetimibe, a phytosterol, a sterol and a stanol. Combinations of IL-10polypeptides with ezetimibe are particularly contemplated herein.Ezetimibe selectively blocks cholesterol absorption and lowers plasmaLDL levels by an average of 18%. When ezetimibe is co-administered withlower doses of statins, there is an additive reduction in LDL levels,which equals the reduction achieved with maximal doses of statins alone.Reduction in the statin dose results in fewer statin-related adverseeffects.

Still further particular embodiments of the present disclosure comprisean IL-10 agent in combination with a fat absorption inhibitor. Fatabsorption inhibitors decrease the absorption of fat from the intestine,thereby reducing caloric intake. In one aspect, a fat absorptioninhibitor inhibits pancreatic lipase, an enzyme that breaks downtriglycerides in the intestine. Examples of suitable fat absorptioninhibitors include, without limitation, Orlistat.

In still other particular embodiments, the present disclosurecontemplates use of the PEG-IL-10 agents described herein in combinationwith modulators of PCSK9 (Proprotein convertase subtilisin/kexin type9). PCSK9 plays a major regulatory role in cholesterol homeostasis. Itis a serine protease expressed predominantly in the liver, intestine andkidney. The encoded protein is synthesized as a soluble zymogen thatundergoes autocatalytic intramolecular processing in the endoplasmicreticulum.

As part of the cholesterol homeostasis process, LDL cholesterol isremoved from the blood when it binds to LDL receptors (LDLR) on thesurface of liver cells and is taken up by such cells. PCSK9 functions bybinding to LDLR and inducing receptor degradation, thereby preventingLDLR recycling to the cell surface to remove more LDL cholesterol,ultimately resulting in decreased metabolism thereof. Preventing PCSK9binding to LDLR allows the receptor to return to the cell surface andremove more cholesterol.

Inhibitors of PCSK9 function have been shown to cause much morecholesterol lowering than traditional commercially available agents,with an acceptable adverse effect profile. The present disclosurecontemplates the use of PEG-IL-10 with any modulator having a direct orindirect inhibitory effect on PCSK9. Several monoclonal antibodies thatbind to PCSK9 and interfere with its interaction with the LDLR are beingdeveloped (e.g., by Amgen (AMG145), Merck (1D05-IgG2) andAventis/Regeneron (SAR236553/REGN727)). In addition, peptides that mimicthe EGFA domain of the LDLR that binds to PCSK9 are being developed, andgene silencing through the administration of a PCSK9 antisenseoligonucleotide (ISIS Pharmaceuticals) has been shown to increaseexpression of the LDLR and decrease circulating total cholesterol levelsin mice. Other modulators of PCSK9 function contemplated for combinationtherapy with the PEG-IL-10 agents described herein are those which actby means of RNA interference (RNAi) (Alnylam Pharmaceuticals) and as alocked nucleic acid (LNA) (Santaris Pharma), also referred to asinaccessible RNA.

The present disclosure encompasses pharmaceutically acceptable salts,acids or derivatives of any of the above.

Immune and Inflammatory Conditions.

The present disclosure provides methods for treating and/or preventingimmune- and/or inflammatory-related diseases, disorders and conditions,as well as disorders associated therewith, with an IL-10 polypeptide(e.g., PEG-IL-10) and at least one additional agent having immune-and/or inflammatory-related properties. By way of example, an IL-10polypeptide may be administered with an agent having efficacy in acardiovascular disorder having an inflammatory component.

Examples of therapeutic agents useful in combination therapy include,but are not limited to, non-steroidal anti-inflammatory drugs (NSAIDs).NSAIDs, a large group of therapeutic compounds with analgesic,anti-inflammatory, and anti-pyretic properties, reduce inflammation byblocking cyclooxygenase. Examples of such agents include ibuprofen, andother propionic acid derivatives (alminoprofen, benoxaprofen, bucloxicacid, carprofen, fenbufen, fenoprofen, fluprofen, flurbiprofen,indoprofen, ketoprofen, miroprofen, naproxen, oxaprozin, pirprofen,pranoprofen, suprofen, tiaprofenic acid, and tioxaprofen); acetic acidderivatives (indomethacin, acemetacin, alclofenac, clidanac, diclofenac,fenclofenac, fenclozic acid, fentiazac, fuirofenac, ibufenac, isoxepac,oxpinac, sulindac, tiopinac, tolmetin, zidometacin, and zomepirac);fenamic acid derivatives (flufenamic acid, meclofenamic acid, mefenamicacid, niflumic acid and tolfenamic acid); biphenylcarboxylic acidderivatives (diflunisal and flufenisal); oxicams (isoxicam, piroxicam,sudoxicam and tenoxican); salicylates (acetyl salicylic acid,sulfasalazine); and the pyrazolones (apazone, bezpiperylon, feprazone,mofebutazone, oxyphenbutazone, phenylbutazone).

Other combinations include selective cyclooxygenase-2 (COX-2)inhibitors, selective cyclooxygenase 1 (COX 1) inhibitors, andnon-selective cyclooxygenase (COX) inhibitors. Particular embodiments ofthe present disclosure contemplate the IL-10 polypeptides describedherein (e.g., PEG-IL-10) in combination with a suitable selective COX-2inhibitor(s), such as Celecoxib, Etoricoxib, Firocoxib, Lumiracoxib,Meloxicam, Parecoxib, Rofecoxib, and Valdecoxib.

Other active agents for combination include steroids such asprednisolone, prednisone, methylprednisolone, betamethasone,dexamethasone, or hydrocortisone. Such a combination may be especiallyadvantageous, since one or more side-effects of the steroid can bereduced or even eliminated by decreasing the steroid dose required whentreating patients in combination with the present IL-10 polypeptides.

Additional examples of active agents for combinations for treating, forexample, rheumatoid arthritis include cytokine suppressiveanti-inflammatory drug(s) (CSAIDs); antibodies to or antagonists ofother human cytokines or growth factors, for example, TNF, LT, IL-1β,IL-2, IL-6, IL-7, IL-8, IL-15, IL-16, IL-18, EMAP-II, GM-CSF, FGF, orPDGF.

Particular combinations of active agents may interfere at differentpoints in the autoimmune and subsequent inflammatory cascade, andinclude TNF antagonists like chimeric, humanized or human TNFantibodies, REMICADE, anti-TNF antibody fragments (e.g., CDP870), andsoluble p55 or p75 TNF receptors, derivatives thereof, p75TNFRIgG(ENBREL.) or p55TNFR1gG (LENERCEPT), soluble IL-13 receptor (sIL-13),and also TNFα converting enzyme (TACE) inhibitors; similarly IL-1inhibitors (e.g., Interleukin-1-converting enzyme inhibitors) may beeffective. Other combinations include Interleukin 11, anti-P7s andp-selectin glycoprotein ligand (PSGL). Other examples of agents usefulin combination with the IL-10 polypeptides described herein includeinterferon-β1a (AVONEX); interferon-β1b (BETASERON); copaxone;hyperbaric oxygen; intravenous immunoglobulin; clabribine; andantibodies to or antagonists of other human cytokines or growth factors(e.g., antibodies to CD40 ligand and CD80).

The present disclosure encompasses pharmaceutically acceptable salts,acids or derivatives of any of the above.

Anti-Diabetic and Anti-Obesity Agents.

Some patients requiring pharmacological treatment for acholesterol-related disorder(s) are also taking anti-diabetic and/oranti-obesity agents. The present disclosure contemplates combinationtherapy with numerous anti-diabetic agents (and classes thereof),including 1) insulin, insulin mimetics and agents that entailstimulation of insulin secretion, including sulfonylureas (e.g.,chlorpropamide, tolazamide, acetohexamide, tolbutamide, glyburide,glimepiride, glipizide) and meglitinides (e.g., repaglinide (PRANDIN)and nateglinide (STARLIX)); 2) biguanides (e.g., metformin (GLUCOPHAGE))and other agents that act by promoting glucose utilization, reducinghepatic glucose production and/or diminishing intestinal glucose output;3) alpha-glucosidase inhibitors (e.g., acarbose and miglitol) and otheragents that slow down carbohydrate digestion and consequently absorptionfrom the gut and reduce postprandial hyperglycemia; 4)thiazolidinediones (e.g., rosiglitazone (AVANDIA), troglitazone(REZULIN), pioglitazone (ACTOS), glipizide, balaglitazone,rivoglitazone, netoglitazone, troglitazone, englitazone, ciglitazone,adaglitazone, darglitazone that enhance insulin action (e.g., by insulinsensitization), thus promoting glucose utilization in peripheraltissues; 5) glucagon-like-peptides including DPP-IV inhibitors (e.g.,vildagliptin (GALVUS) and sitagliptin (JANUVIA)) and Glucagon-LikePeptide-1 (GLP-1) and GLP-1 agonists and analogs (e.g., exenatide(BYETTA)); 6) and DPP-IV-resistant analogues (incretin mimetics), PPARgamma agonists, dual-acting PPAR agonists, pan-acting PPAR agonists,PTP1B inhibitors, SGLT inhibitors, insulin secretagogues, glycogensynthase kinase-3 inhibitors, immune modulators, beta-3 adrenergicreceptor agonists, 11beta-HSD1 inhibitors, and amylin analogues. Instill other embodiments, the IL-10 agents described herein are used incombination with one or more suitable nuclear receptor binding agents(e.g., a Retinoic Acid Receptor (RAR) binding agent, a Retinoid XReceptor (RXR) binding agent, a Liver X Receptor (LXR) binding agent anda Vitamin D binding agent).

Furthermore, the present disclosure contemplates combination therapywith agents and methods for promoting weight loss, such as agents thatstimulate metabolism or decrease appetite, and modified diets and/orexercise regimens to promote weight loss.

The present disclosure encompasses pharmaceutically acceptable salts,acids or derivatives of any of the above.

Dosing

The IL-10 polypeptides of the present disclosure may be administered toa subject in an amount that is dependent upon, for example, the goal ofthe administration (e.g., the degree of resolution desired); the age,weight, sex, and health and physical condition of the subject; the routeof administration; and the nature of the disease, disorder, condition orsymptom thereof. The dosing regimen may also take into consideration theexistence, nature, and extent of any adverse effects associated with theagent(s) being administered. Effective dosage amounts and dosageregimens can readily be determined from, for example, safety anddose-escalation trials, in vivo studies (e.g., animal models), and othermethods known to the skilled artisan.

In general, dosing parameters dictate that the dosage amount be lessthan an amount that could be irreversibly toxic to the subject (i.e.,the maximum tolerated dose, “MTD”) and not less than an amount requiredto produce a measurable effect on the subject. Such amounts aredetermined by, for example, the pharmacokinetic and pharmacodynamicparameters associated with ADME, taking into consideration the route ofadministration and other factors.

An effective dose (ED) is the dose or amount of an agent that produces atherapeutic response or desired effect in some fraction of the subjectstaking it. The “median effective dose” or ED50 of an agent is the doseor amount of an agent that produces a therapeutic response or desiredeffect in 50% of the population to which it is administered. Althoughthe ED50 is commonly used as a measure of reasonable expectance of anagent's effect, it is not necessarily the dose that a clinician mightdeem appropriate taking into consideration all relevant factors. Thus,in some situations the effective amount is more than the calculatedED50, in other situations the effective amount is less than thecalculated ED50, and in still other situations the effective amount isthe same as the calculated ED50.

In addition, an effective dose of the IL-10 polypeptides of the presentdisclosure may be an amount that, when administered in one or more dosesto a subject, produces a desired result relative to a healthy subject.For example, for a subject experiencing a particular disorder, aneffective dose may be one that improves a diagnostic parameter, measure,marker and the like of that disorder by at least about 5%, at leastabout 10%, at least about 20%, at least about 25%, at least about 30%,at least about 40%, at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, or more than 90%,where 100% is defined as the diagnostic parameter, measure, marker andthe like exhibited by a normal subject.

When an IL-10 polypeptide is PEG-IL-10, the amount of PEG-IL-10necessary to treat a disease, disorder or condition described herein isbased on the IL-10 activity of the conjugated protein, which, asindicated above, can be determined by IL-10 activity assays known in theart. By way of example, in the tumor context, suitable IL-10 activityincludes, for example, CD8+ T-cell infiltrate into tumor sites,expression of inflammatory cytokines, such as IFN-γ, IL-4, IL-6, IL-10,and RANK-L, from these infiltrating cells, and increased levels of TNF-αor IFN-γ in biological samples.

Like many drugs, intravenous IL-10 administration is associated with atwo-compartment kinetic model (see Rachmawati, H. et al. (2004) Pharm.Res. 21(11):2072-78). Plasma drug concentrations decline in amulti-exponential fashion. Immediately after intravenous administration,the drug rapidly distributes throughout an initial space (minimallydefined as the plasma volume), and then a slower, equilibrativedistribution to extravascular spaces (e.g., certain tissues) occurs. Thepharmacokinetics of subcutaneous recombinant hIL-10 has also beenstudied (Radwanski, E. et al. (1998) Pharm. Res. 15(12):1895-1901).Volume-of-distribution and other pharmacokinetic considerations arepertinent when assessing appropriate IL-10 dosing-related parameters.Moreover, the leveraging of IL-10 pharmacokinetic and dosing principlesmay prove invaluable to the success of efforts to target IL-10 agents tospecific cell types (see, e.g., Rachmawati, H. (May 2007) Drug Met.Dist. 35(5):814-21).

The present disclosure contemplates administration of any dose anddosing regimen that results in the desired therapeutic outcome. By wayof example, but not limitation, when the subject is a human,non-pegylated hIL-10 may be administered at a dose greater than 0.5μg/kg/day, greater than 1.0 μg/kg/day, greater than 2.5 μg/kg/day,greater than 5 μg/kg/day, greater than 7.5 μg/kg, greater than 10.0μg/kg, greater than 12.5 μg/kg, greater than 15 μg/kg/day, greater than17.5 μg/kg/day, greater than 20 μg/kg/day, greater than 22.5 μg/kg/day,greater than 25 μg/kg/day, greater than 30 μg/kg/day, or greater than 35μg/kg/day. In addition, by way of example, but not limitation, when thesubject is a human, pegylated hIL-10 comprising a relatively small PEG(e.g., 5 kDa mono-di-PEG-hIL-10) may be administered at a dose greaterthan 0.5 μg/kg/day, greater than 0.75 μg/kg/day, greater than 1.0μg/kg/day, greater than 1.25 μg/kg/day, greater than 1.5 μg/kg/day,greater than 1.75 μg/kg/day, greater than 2.0 μg/kg/day, greater than2.25 μg/kg/day, greater than 2.5 μg/kg/day, greater than 2.75 μg/kg/day,greater than 3.0 μg/kg/day, greater than 3.25 μg/kg/day, greater than3.5 μg/kg/day, greater than 3.75 μg/kg/day, greater than 4.0 μg/kg/day,greater than 4.25 μg/kg/day, greater than 4.5 μg/kg/day, greater than4.75 μg/kg/day, or greater than 5.0 μg/kg/day.

The therapeutically effective amount of PEG-IL-10 can range from about0.01 to about 100 μg protein/kg of body weight/day, from about 0.1 to 20μg protein/kg of body weight/day, from about 0.5 to 10 μg protein/kg ofbody weight/day, or from about 1 to 4 μg protein/kg of body weight/day.In some embodiments, PEG-IL-10 is administered by continuous infusion todelivery about 50 to 800 μg protein/kg of body weight/day (e.g., about 1to 16 μg protein/kg of body weight/day of PEG-IL-10). The infusion ratemay be varied based on evaluation of, for example, adverse effects andblood cell counts.

For administration of an oral agent, the compositions can be provided inthe form of tablets, capsules and the like containing from 1.0 to 1000milligrams of the active ingredient, particularly 1.0, 3.0, 5.0, 10.0,15.0, 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0,500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the activeingredient.

In certain embodiments, the dosage of the disclosed IL-10 polypeptide(e.g., PEG-IL-10) is contained in a “unit dosage form”. The phrase “unitdosage form” refers to physically discrete units, each unit containing apredetermined amount of a IL-10 polypeptide of the present disclosure,either alone or in combination with one or more additional agents,sufficient to produce the desired effect. It will be appreciated thatthe parameters of a unit dosage form will depend on the particular agentand the effect to be achieved.

Kits

The present disclosure also contemplates kits comprising IL-10polypeptides (e.g., PEG-IL-10), and pharmaceutical compositions thereof.The kits are generally in the form of a physical structure housingvarious components, as described below, and may be utilized, forexample, in practicing the methods described above (e.g., administrationof an IL-10 polypeptide to a subject in need of restoring cholesterolhomeostasis).

A kit can include one or more of the IL-10 polypeptides disclosed herein(provided in, e.g., a sterile container), which may be in the form of apharmaceutical composition suitable for administration to a subject. TheIL-10 polypeptides can be provided in a form that is ready for use or ina form requiring, for example, reconstitution or dilution prior toadministration. When the IL-10 polypeptides are in a form that needs tobe reconstituted by a user, the kit may also include buffers,pharmaceutically acceptable excipients, and the like, packaged with orseparately from the IL-10 polypeptides. When combination therapy iscontemplated, the kit may contain the several agents separately or theymay already be combined in the kit. Each component of the kit may beenclosed within an individual container, and all of the variouscontainers may be within a single package. A kit of the presentdisclosure may be designed for conditions necessary to properly maintainthe components housed therein (e.g., refrigeration or freezing).

A kit may contain a label or packaging insert including identifyinginformation for the components therein and instructions for their use(e.g., dosing parameters, clinical pharmacology of the activeingredient(s), including mechanism of action, pharmacokinetics andpharmacodynamics, adverse effects, contraindications, etc.). Labels orinserts can include manufacturer information such as lot numbers andexpiration dates. The label or packaging insert may be, e.g., integratedinto the physical structure housing the components, contained separatelywithin the physical structure, or affixed to a component of the kit(e.g., an ampule, tube or vial).

Labels or inserts can additionally include, or be incorporated into, acomputer readable medium, such as a disk (e.g., hard disk, card, memorydisk), optical disk such as CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape,or an electrical storage media such as RAM and ROM or hybrids of thesesuch as magnetic/optical storage media, FLASH media or memory-typecards. In some embodiments, the actual instructions are not present inthe kit, but means for obtaining the instructions from a remote source,e.g., via the internet, are provided.

EXPERIMENTAL

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below were performed and areall of the experiments that may be performed. It is to be understoodthat exemplary descriptions written in the present tense were notnecessarily performed, but rather that the descriptions can be performedto generate the data and the like described therein. Efforts have beenmade to ensure accuracy with respect to numbers used (e.g., amounts,temperature, etc.), but some experimental errors and deviations shouldbe accounted for.

Unless indicated otherwise, parts are parts by weight, molecular weightis weight average molecular weight, temperature is in degrees Celsius (°C.), and pressure is at or near atmospheric. Standard abbreviations areused, including the following: bp=base pair(s); kb=kilobase(s);pl=picoliter(s); s or sec=second(s); min=minute(s); h or hr=hour(s);aa=amino acid(s); kb=kilobase(s); nt=nucleotide(s); pg=picogram;ng=nanogram; μg=microgram; mg=milligram; g=gram; kg=kilogram; dl ordL=deciliter; μl or μL=microliter; ml or mL=milliliter; 1 or L=liter;μM=micromolar; mM=millimolar; M=molar; kDa=kilodalton;i.m.=intramuscular(ly); i.p.=intraperitoneal(ly); SC orSQ=subcutaneous(ly); QD=daily; BID=twice daily; QW=weekly; QM=monthly;wt=wildtype; HPLC=high performance liquid chromatography; BW=bodyweight; U=unit; ns=not statistically significant; PBS=phosphate-bufferedsaline; PCR=polymerase chain reaction; NHS=N-Hydroxysuccinimide;Cl=clodronate; HSA=human serum albumin; MSA=mouse serum albumin;IHC=immunohistochemistry; DMEM=Dulbeco's Modification of Eagle's Medium;GC=genome copy; EDTA=ethylenediaminetetraacetic acid; PCSK9=Proproteinconvertase subtilisin/kexin type 9; CYP7A1=cytochrome P450 7A1,cholesterol 7 alpha-hydroxylase, or cholesterol 7-alpha-monooxygenase;ABCG1=ATP-binding cassette sub-family G member 1; MSR1=MacrophageScavenger Receptor 1.

Materials and Methods

The following general materials and methods were used, where indicated,or may be used in the Examples below:

Standard methods in molecular biology are described in the scientificliterature (see, e.g., Sambrook and Russell (2001) Molecular Cloning,3^(rd)ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.; and Ausubel, et al. (2001) Current Protocols in Molecular Biology,Vols. 1-4, John Wiley and Sons, Inc. New York, N.Y., which describescloning in bacterial cells and DNA mutagenesis (Vol. 1), cloning inmammalian cells and yeast (Vol. 2), glycoconjugates and proteinexpression (Vol. 3), and bioinformatics (Vol. 4)).

The scientific literature describes methods for protein purification,including immunoprecipitation, chromatography, electrophoresis,centrifugation, and crystallization, as well as chemical analysis,chemical modification, post-translational modification, production offusion proteins, and glycosylation of proteins (see, e.g., Coligan, etal. (2000) Current Protocols in Protein Science, Vols. 1-2, John Wileyand Sons, Inc., N.Y.).

Production, purification, and fragmentation of polyclonal and monoclonalantibodies are described (e.g., Harlow and Lane (1999) Using Antibodies,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.); standardtechniques for characterizing ligand/receptor interactions are available(see, e.g., Coligan et al. (2001) Current Protocols in Immunology, Vol.4, John Wiley, Inc., N.Y.); methods for flow cytometry, includingfluorescence-activated cell sorting (FACS), are available (see, e.g.,Shapiro (2003) Practical Flow Cytometry, John Wiley and Sons, Hoboken,N.J.); and fluorescent reagents suitable for modifying nucleic acids,including nucleic acid primers and probes, polypeptides, and antibodies,for use, for example, as diagnostic reagents, are available (MolecularProbes (2003) Catalogue, Molecular Probes, Inc., Eugene, Oreg.;Sigma-Aldrich (2003) Catalogue, St. Louis, Mo.).

Software packages and databases for determining, e.g., antigenicfragments, leader sequences, protein folding, functional domains,glycosylation sites, and sequence alignments, are available (see, e.g.,GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.); and DeCypher™(TimeLogic Corp., Crystal Bay, Nev.).

Normalized mRNA units may be determined by relative quantificationwhereby changes in gene expression in a sample are calculated based ongene expression in a reference sample.

Mice:

LDLR−/− mice were obtained from The Jackson Lab (Bar Harbor, Me.).LDLR−/− mice have an elevated serum cholesterol level of 200-400 mg/dl,and their cholesterol level averages ˜1,500 mg/dL when fed a high fatdiet. Normal serum cholesterol in the mouse is 80-100 mg/dl.

Serum IL-10 Concentrations:

For the experiments described herein, concentrations of test materialsin serum samples were determined via an electrochemiluminescence assayfor mIL-10 on the MDS platform. LCB-33AEY (ARMO) anti-MIL-10 was used asthe capture and biotinylated anti-MIL-10 (R&D) was used as the detectionantibody. A PEG-rMuIL-10 standard curve was set up with the upper andlower limits of 195,000 pg-9 pg. Plates were incubated for 2 hours atroom temperature, and after washing detected via the SulfoTag program onan MSD 2400 platform.

Alternatively, serum IL-10 concentration levels and exposure levels maybe determined by other standard methods used in the art. For example, aserum exposure level assay can be performed by collecting whole blood(˜50 μl/mouse) from mouse tail snips into plain capillary tubes,separating serum and blood cells by centrifugation, and determiningIL-10 exposure levels by standard ELISA kits and techniques.

Serum and Tissue Cholesterol Quantitation:

Methodology No. 1:

Serum was analyzed at a 1:300 dilution. Mouse livers were ground up in200 μL water with pestles (VWR). Twenty-five microliter aliquots wereused for the extraction of cholesterol/cholesteryl esters using acholesterol/cholesteryl assay kit (BioVision) according to themanufacturer's instructions and measured with a Spectra Max 340 PC(Molecular Devices). Genomic DNA was extracted from equal amounts ofsample using a DNeasy kit (QIAGEN) and measured with a NanoVue Plus (GEHealthcare) to normalize the cholesterol/cholesteryl measurements.

Methodology No. 2:

Serum LDL-C or HDL-C quantitation was performed on a Beckman Coulter AUSystem LDL-Cholesterol test using a two reagent homogenous system. Theassay is comprised of two distinct phases. In Phase 1, a uniquedetergent solubilizes cholesterol from non-LDL-lipoprotein ornon-HDL-particles, depending on the desired assay output. Thischolesterol is consumed by cholesterol esterase, cholesterol oxidase,peroxides, and 4 aminoantipyrine (LDL-C) or DSBmT (HDL-C) to generate acolorless end product. In Phase 2, a second detergent in the R2 reagentreleases cholesterol from the remaining lipoproteins. This cholesterolreacts with cholesterol esterase, cholesterol oxidase, and a chromogensystem to yield a blue color complex which can be measuredbichromatically at 540/660 nm. The resulting increase in absorbance isdirectly proportional to the LDL-C or HDL-C concentration in the sample.

qPCR Analysis:

Livers were excised from mice at necropsy and flash-frozen forsubsequent analysis. Mouse livers were ground up in Buffer RLT (QIAGEN)with 10 μL β-mercaptoethanol (Sigma-Aldrich) using pestles (VWR), afterwhich RNA was extracted using an RNeasy kit (QIAGEN) according to themanufacturer's instructions. The purified RNA was used as template forRT-PCR using an RT2 First Strand kit (QIAGEN). One microliter aliquotsof the resulting cDNA samples were used for qPCR of the indicatedtranscripts on an ABI PRISM 7700 Sequence Detection System or an ABIViiA 7 Real Time PCR Machine (Life Technologies). CT values werenormalized to the average CT value of Gapdh and Gusb.

Tissue Triglyceride Quantitation:

Mouse livers were ground up in 200 μL water with pestles (VWR).Twenty-five microliter aliquots were used for the extraction oftriglycerides using a Triglyceride Assay kit (Biovision) according tothe manufacturer's instructions and measured with a Spectra Max 340 PC(Molecular Devices). Protein concentration was determined using thePierce BCA protein assay (Thermo Scientific) according to themanufacturer's instructions and measured with a Spectra Max 340 PC.

Immunohistochemistry:

Picosirius Red Stain:

Slides were heated in an oven at 60° C. for 45 mins, de-paraffinizedusing xylene and series of alcohols, and rehydrated in water; then keptfor 60 mins in freshly-prepared Picosirius red solution according tomanufacturer's instructions, followed by two washes in acidified water.Nuclei were stained with Weigert's hematoxylin for 8-10 mins, dehydratedin three changes of 100% ethanol, cleared, and mounted.

Hematoxylin and Eosin Stain:

Slides were de-paraffinized with two changes of xylene (10 mins each),dehydrated through absolute, 95%, 70% alcohols (5 mins each), andrehydrated in water. Slides were then placed in hematoxylin stain for 4mins, rinsed in water, differentiated in 1% acid alcohol for 30 seconds,and stained in 0.01% Eosin Y for 2 sec, rinsed in 95% ethanol,dehydrated with absolute ethanol, and cleared in xylenes for 15 minsbefore cover slipping.

Anti-F4/80, Anti-Msr1, Anti-PCNA:

Liver tissues were fixed with 10% neutral-buffered formaldehyde and wereembedded in paraffin. Tissue specimens were cut into 5-μm-thicksections, de-paraffinized in xylene sections, and hydrated in a gradedseries of alcohol solutions (100%, 95%, 80%, 70%, 50% (three changes,each of 5 mins)). The tissues on slides underwent heat-induced epitoperetrieval (10 mmol/L sodium citrate buffer at 98° C. for 20 mins), thenwere treated with 3% H₂O₂ to quench endogenous peroxidase. Sections wereincubated in blocking solution (5% neutral goat serum) for 1 hr at roomtemperature. Primary antibodies of choice were applied on the slides andincubated in humid chamber overnight at 4° C. Secondary biotinylatedantibody was then applied at 1:250 dilution (Vector Lab, Burlingame,Calif., USA), followed by incubation with streptavidin peroxidase.Sections were washed with PBS three times after each step. Sections werestained with DAB substrate and counterstained with Mayer's hematoxylinfor 2 mins. Slides were dehydrated in three changes of 100% ethanol,cleared, and mounted.

Image Quantitation:

For analysis of PEG-rMuIL-10-treated livers compared to vehicle-treatedlivers, 5-10 mice per group were randomly selected and stained withSirius Red (Polyscience Inc.), TUNEL (Roche), anti-PCNA (Abcam),Hematoxylin (American MasterTech), anti-Msr1 (Abcam), anti-F4/80(Abcam), or anti-CD68 (Serotech). For each liver, 8-10 independentimages were collected using the 20×objective. An average area of signalwas then analyzed using MetaMorph Imaging Software (Molecular Devices)by applying a color threshold on a representative field and adjustingthe pixel distribution to correspond with a positive signal.

In Vitro Uptake Assays:

Monocytes were isolated from Ficoll centrifugation-isolated peripheralblood mononuclear cells by Miltenyi magnetic bead positive selection.Cells were stimulated with or without PEG-rHuIL-10 for 24 hours incomplete RPMI. 1×10⁶ cells were analyzed for uptake of 20 μL DiLDL,OxLDL or AcLDL, (Alfa Aesar) in 4 hrs. Macrophages were differentiatedfrom positively selected peripheral blood monocytes with 50 ng/mL M-CSF(BioLegend) in cRPMI for 7 days. Cells were re-plated at 0.3-0.4×10⁶cells per well in a 24-well plate and exposed to PEG-rHuIL-10 for 24hrs. Cells were washed once and exposed to 20 μL DiLDL, OxLDL or AcLDL,(Alfa Aesar), where uptake was measured after 4 hrs. Human primaryhepatocytes (Triangle Research Labs) and Kupffer cells (Invitrogen) werethawed and plated at 0.4×10⁶ cells per well in 24-well plates andincubated overnight in hepatocyte incubation medium (phenol-red freeRPMI, pen/strep, Cell Maintenance Supplement B (Invitrogen)) andcomplete RPMI, respectively. Cells were washed and exposed for 24 hrs toPEG-rHuIL-10. Thereafter, cells were washed once and exposed to 20 μLDiLDL, OxLDL or AcLDL, (Alfa Aesar), where uptake was measured after 4hrs. All cells were washed once in 1×PBS and lysed with 110 μL celllysis buffer (Sigma-Aldrich). Forty-five μL of cell lysis wastransferred to clear bottom black-walled plates (Greiner Bio-One) wherefluorescence was read at 575 nm.

In Vivo Studies:

Mouse studies were conducted at Aragen Biosciences (Morgan Hill, Calif.)in accordance with standard operating procedures and establishedguidelines approved by their Institutional Animal Care and Use Committee(IACUC).

Hypercholesterolemia Model:

In-life portions of the studies were performed at Aragen Biosciences.Wild-type and LDLR−/− C57BL/6 mice (7-8 weeks old) were maintained onnormal chow or fed a Western diet for 2 or 7 weeks prior to dosing.PEGylated recombinant mouse IL-10 (PEG-rMuIL-10) or vehicle (10 mMHEPES, 100 mM NaCl, pH 6.5, 0.05% mouse serum albumin) was dosed SCdaily for 2 to 3 weeks; mice were maintained on their respective dietsthroughout dosing. For clodronate depletion studies, mice were dosed IVevery 3 days with clodronate liposomes (5 mg/mL clodronate) or vehicleliposomes (first dose: 0.2 mL, subsequent doses: 0.1 mL), starting oneday before PEG-rMuIL-10 dosing.

Pegylation:

A mono-/di-PEG-IL-10 mix described in the patent literature (e.g., US2011/0250163) was used in both pre-clinical and clinical analyses, andin the examples set forth hereafter. Two exemplary synthetic schemes areas follows:

Exemplary Scheme No. 1.

IL-10 (e.g., rodent or primate) is dialyzed against 50 mM sodiumphosphate, 100 mM sodium chloride pH ranges 5-7.4. A 1:1-1:7 molar ratioof 5 kDa PEG-propyladehyde is reacted with IL-10 at a concentration of1-12 mg/mL in the presence of 0.75-30 mM sodium cyanoborohydride.Alternatively the reaction can be activated with picoline borane in asimilar manner. The reaction is incubated at 5-30° C. for 3-24 hours.The pH of the pegylation reaction is adjusted to 6.3, and 7.5 mg/mL ofhIL-10 is reacted with PEG to make the ratio of IL-10 to PEG linker1:3.5. The final concentration of cyanoborohydride is ˜25 mM, and thereaction is carried out at 15° C. for 12-15 hours. The mono- and di-PEGIL-10 are the largest products of the reaction, with the concentrationof each at ˜50% at termination. The reaction may be quenched using anamino acid such as glycine or lysine or, alternatively, Tris buffers.Multiple purification methods can be employed such as gel filtration,anion and cation exchange chromatographies, and size exclusion HPLC(SE-HPLC) to isolate the desired pegylated IL-10 molecules.

Exemplary Scheme No. 2.

IL-10 is dialyzed against 10 mM sodium phosphate pH 7.0, 100 mM NaCl.The dialyzed IL-10 is diluted 3.2 times to a concentration of about 0.5to 12 mg/mL using the dialysis buffer. Prior to the addition of thelinker, SC-PEG-12 kDa (Delmar Scientific Laboratories, Maywood, Ill.)and one volume of 100 mM Na-tetraborate at pH 9.1 is added into 9volumes of the diluted IL-10 to raise the pH of the IL-10 solution to8.6. The SC-PEG-12K linker is dissolved in the dialysis buffer and theappropriate volume of the linker solution (1.8 to 3.6 mole linker permole of IL-10) is added into the diluted IL-10 solution to initiate thepegylation reaction. The reaction is carried out at 5° C. in order tocontrol the rate, and the reaction solution is mildly agitated. When themono-PEG-IL-10 yield, as determined by size exclusion HPLC (SE-HPLC), isclose to 40%, the reaction is stopped by adding 1M glycine solution to afinal concentration of 30 mM. The pH of the reaction solution is slowlyadjusted to 7.0 using an HCl solution, and the reaction is 0.2 micronfiltered and stored at −80° C.

PEG-IL-10 was formulated at 0.75-1.0 mg/mL in 10 mM HEPES, pH 6.5, 100mM NaCl containing 0.05% MSA. Control (placebo)=same formulation matrixwithout PEG-IL-10.

EXAMPLE 1 PEG-rMuIL-10 Lowers Plasma Cholesterol Levels

The regulatory effect of PEG-rMuIL-10 on plasma cholesterol levels inwild-type and LDLR−/− mice fed a normal and high fat diet was assessed.

Referring to FIGS. 1A-1C, wild-type mice on normal chow exhibited nodecrease in total plasma cholesterol, LDL or HDL particles followingadministration of 1.0, 0.2, and 0.02 mg/kg PEG-rMuIL-10, or vehiclecontrol, SC daily.

The effect of PEG-rMuIL-10 in LDLR−/− mice was then determined. Whentotal plasma cholesterols levels reached approximately 200 mg/dL in bothLDLR−/− mice on normal chow diet and wild-type mice on high-fat chowdiet, 0.2 mg/kg PEG-rMuIL-10, 0.02 mg/kg PEG-rMuIL-10, or vehiclecontrol were administered SC. Mice that received 0.2 mg/kg PEG-rMuIL-10exhibited cholesterol reduction of 22% (FIG. 1D; LDLR−/− mice) and 25%(FIG. 1G; wt mice). Mice that received 0.2 mg/kg PEG-rMuIL-10 exhibitedLDL-C reduction of 30% (FIG. 1E; LDLR−/− mice) and 15% (FIG. 1H; wtmice). Mice that received 0.2 mg/kg PEG-rMuIL-10 exhibited HDL-Creduction of 36% (FIG. 1F; LDLR−/− mice) and 45% (FIG. 1I; wt mice).LDLR−/− mice on a high fat chow diet that received 0.02 mg/kgPEG-rMuIL-10, SC daily for two weeks exhibited a total plasmacholesterol level reduction of nearly 50%, an LDL-C level reduction of60%, and an HDL-C reduction of 35% (FIGS. 1J-L, respectively) wheninitial levels of cholesterol and LDL-C were higher. FIGS. 1A-1L alsoindicates the effect of 0.02 mg/kg PEG-rMuIL-10 on each of theaforementioned parameters. Lastly, longer dosing of the LDLR−/− mice ona high fat chow diet resulted in total plasma cholesterol reduction ofup to 70% (data not show). Taken together, the results indicate that thelowering of plasma cholesterol with PEG-rMuIL-10 is dependent on totalcholesterol levels.

In order to confirm the findings set forth above, total plasmacholesterol was assessed in human patients (n=4) being treated forcancer (a mix of non-small cell lung carcinoma (NSCLC), renal cellcarcinoma (RCC) and colorectal) with PEG-rHuIL-10. Patients wereadministered 2.5 μg/kg or 5 μg/kg PEG-rHuIL-10 SC daily. As indicated inFIG. 1M, administration of 2.5 μg/kg PEG-rHuIL-10 lowered cholesterolthe most in patients with borderline-high (˜200 mg/dL) totalcholesterol; in this patient population, total plasma cholesterol wasreduced by up to 40%, consistent with the mouse data discussedpreviously. Patients with low (˜100 mg/dL) total cholesterol wereunaffected by PEG-rHuIL-10, and patients with an initial plasmacholesterol concentration of ˜140 mg/dL achieved cholesterol lowering toabout 100 mg/dL. A level of 140 mg/dL or lower is consistent with nolife time risk of a cardiovascular event. Administration of 5 μg/kgPEG-rHuIL-10 resulted in cholesterol reduction in a manner consistentwith that observed when 2.5 μg/kg PEG-rHuIL-10 was adminstered, and theamount of reduction was also dependent on the patient's baselinecholesterol level (see FIG. 1N).

These data suggest that the mechanism by which PEG-IL-10 regulates serumcholesterol in mice is similar to that in humans.

EXAMPLE 2 PEG-rMuIL-10 Alters Liver Expression of Cholesterol Synthesisand Scavenger Receptor Genes

Using the Qiagen Lipoprotein Signaling and Cholesterol Metabolism,Fibrosis and Innate & Adaptive Immune Response Panels, the effect ofPEG-rMuIL-10 on the expression of genes associated with liver functionand cholesterol regulation was evaluated. As illustrated in FIGS. 2A-2L,only two primary groups of genes were altered in response toPEG-rMuIL-10 exposure.

The first group of regulatory genes is involved in endogenouscholesterol synthesis. Wild-type mice on normal chow diet (FIG. 2A),LDLR−/− mice on normal chow diet (FIG. 2B), wild-type mice on high-fatchow diet (FIG. 2C), and LDLR−/− mice on high-fat chow diet (FIG. 2D),were administered 0.2 mg/kg PEG-rMuIL-10, 0.02 mg/kg PEG-rMuIL-10, orvehicle control, SC daily. Hmgcs1 and 2, two genes involved inendogenous cholesterol synthesis, were moderately but consistentlydecreased in the liver (FIGS. 2A-D). These data are consistent withreports that IL-10 may decrease endogenous cholesterol synthesis byinhibiting Mevalonate Pathway.

The second group of regulatory genes comprises scavenger receptors. Asindicated in FIGS. 2E-2L, all further comparisons focused on thedifferences between the vehicle control group and the group of miceadministered 0.2 mg/kg PEG-rMuIL-10, as the group of mice administered0.02 mg/kg PEG-rMuIL-10 exerted limited control on both plasmacholesterol control and gene regulation. Wild-type mice on normal chowdiet (FIG. 2E), LDLR−/− mice on normal chow diet (FIG. 2F), wild-typemice on high-fat chow diet (FIG. 2G), and LDLR−/− mice on high-fat chowdiet (FIG. 2H), were administered 0.2 mg/kg PEG-rMuIL-10 or vehiclecontrol, SC daily. As indicated in FIGS. 2E-2H, Msr1 and Marco, Type Ascavenger receptors, were induced by 2-7 fold. In order to expand thescavenger receptor analysis, expression differences in other receptorsknown to regulate serum cholesterol were assessed. LDL Receptorexpression was not changed (data not shown) and is not relevant in theLDLR−/−, and expression of both CD36 and PCSK9 were also unchanged (seeFIGS. 2E-2H). Because scavenger receptors are predominantly expressed onmacrophage-type cells, differences in expression of F4/80 and CD14, twocell surface proteins most often expressed on liver tissue residentmacrophages, were also assessed. These genes were moderately inducedacross the different genetic backgrounds and dietary conditions (seeFIGS. 2E-2H). These data confirm the role of scavenger receptors inaspects of liver function and cholesterol regulation.

If PEG-rMuIL-10 induces an increase in the uptake of lipoproteins, theremay be a concomitant increase in efflux-associated genes. Therefore, thelevels of the efflux mediators ABCG1 and ABCA1 were evaluated. ABCG1 isconsidered to principally function in the liver, and ABCA1 is consideredto primarily function in the gastrointestinal track. Both factors act ascholesterol efflux pumps to remove cholesterol from the interior of thecell onto lipid-poor HDL particles. Wild-type mice on normal chow diet(FIG. 2I), LDLR−/− mice on normal chow diet (FIG. 2J), wild-type mice onhigh-fat chow diet (FIG. 2K), and LDLR−/− mice on high-fat chow diet(FIG. 2L), were administered 0.2 mg/kg PEG-rMuIL-10, or vehicle control,SC daily. As depicted in FIGS. 2I-L, only ABCG1 exhibits a consistent,though moderate, trend of increased expression.

EXAMPLE 3 PEG-rMuIL-10 Alters Expression of Scavenger Receptors and thePresence of Kupffer Cells

In order to evaluate whether PEG-rMuIL-10 increases both scavengerreceptors and the number of KCs, treated and untreated liver tissue wasanalyzed for differences in MSR1 expression by IHC.

Wild-type mice on normal chow diet (FIG. 3A), LDLR−/− mice on normalchow diet (FIG. 3B), wild-type mice on high-fat chow diet (FIG. 3C), andLDLR−/− mice on high-fat chow diet (FIG. 3D), were administered 0.2mg/kg PEG-rMuIL-10 or vehicle control, SC daily. As indicated in FIGS.3A and 3B, dosing with PEG-rMuIL-10 led to a slight increase indetectible MSR1 in wt and LDLR−/− mice on the normal diet. However, MSR1levels in control tissue appeared to decrease in conjunction withincreased dietary fat intake. The levels of detectible MSR1 were higherin wt and LDLR−/− mice on normal chow relative to the same type of miceon high fat chow. This result was confirmed with the use of IHC toquantify the amount of Msr1 (FIGS. 3A-D). In addition, treatment withPEG-rMuIL-10 appeared to normalize levels of Msr1 within the livertissue of LDLR−/− mice (data not shown).

Referring to FIGS. 2A-2L, scavenger receptors are most often expressedon macrophage lineage cells and an increase in message expression ofF4/80 and CD14 was identified. Thus, wild-type mice on normal chow diet(FIG. 3E), LDLR−/− mice on normal chow diet (FIG. 3F), wild-type mice onhigh-fat chow diet (FIG. 3G), and LDLR−/− mice on high-fat chow diet(FIG. 3H) were administered 0.2 mg/kg PEG-rMuIL-10 or vehicle control,SC daily in order to assess the protein expression levels of F4/80 byIHC (data not shown). In wild type and LDLR−/− mice on normal chow,there was little-to-no difference in the presence of F4/80-positivecells (FIGS. 3E and F). However, in wild type and LDLR−/− on high fatchow, there was a detectible increase in F4/80-positive cells (FIGS. 3Gand H), suggesting PEG-rMuIL-10 increases the number of Kupffer cells inthe liver of animals with increased dietary fat uptake.

In addition, expression analysis of the IL-10Rα showed an approximate2-fold difference in IL-10Rα message between wt and LDLR−/− mice onnormal vs. high fat chow, suggesting that some of the difference incholesterol regulation may be due to relative IL-10 receptor levels(data not shown).

EXAMPLE 4 In Vivo Depletion of Phagocytotic Cells Abolishes PEG-rMuIL-10Reduction of Cholesterol

An assessment was conducted in order to determine whether cellsassociated with the myeloid lineage were responsible for the control ofplasma cholesterol by PEG-rMuIL-10.

A standard technique was used for removing phagocytotic cells by dosinganimals with clodronate (Cl) liposomes in the presence or absence ofPEG-rMuIL-10. After ensuring complete depletion of all phagocytoticcells in the liver and assessing hepatocyte health by IHC (data notshown), PEG-rMuIL-10 regulation of plasma cholesterol levels wasevaluated in wild-type mice on normal chow diet (FIG. 4B), LDLR−/− miceon normal chow diet (FIG. 4C), wild-type mice on high-fat chow diet(FIG. 4D), and LDLR−/− mice on high-fat chow diet (FIG. 4A).

As indicated in FIGS. 4A-4G, depletion of phagocytotic cells abolishedthe regulation of PEG-rMuIL-10 of plasma cholesterol. PEG-rMuIL-10-dosedmice (1 mg/kg) exhibited a 50% decrease in cholesterol, while mice dosedwith both PEG-rMuIL-10 and clodronate liposome had a 25% increase (FIG.4A). Unexpectedly, the mice that received only clodronate exhibited a45% increase in total plasma cholesterol. Thereafter, phagocytotic cellswere depleted in wild type and LDLR−/− mice on normal chow diet (FIGS.4B and 4C, respectively), and wt mice on high fat chow diet (FIG. 4D.Surprisingly, removal of phagocytotic cells consistently increasedplasma cholesterol, suggesting phagocytotic cells in general areimplicit in the normal regulation of plasma cholesterol.

IHC assessment indicated that PEG-rMuIL-10 exerted no effect onclodronate-mediated depletion of liver Kupffer cells at day 7, but thatPEG-rMuIL-10 had begun to facilitate the repopulation of Kupffer cellsin the liver by day 14 (data not shown). As indicated in FIG. 4E,PEG-rMuIL-10's repopulation of liver Kupffer cells is consistent with adecrease in plasma cholesterol.

Dosing with PEG-rMuIL-10 appeared to enhance the entry of monocytes fromthe bone marrow into the systemic circulation. As indicated in FIGS. 4Fand 4G, there is an initial decrease of peripheral monocytes of miceafter exposure to clodronate. PEG-rMuIL-10 then acted to facilitate therecovery of this cell population. These data suggest that dosing withPEG-rMuIL-10 may slowly restore homeostasis, overcoming the effects ofclodronate and enhancing the repopulation of Kupffer cells in the liver,thereby reestablishing their ability to lower cholesterol.

EXAMPLE 5 PEG-rHuIL-10 Induces Scavenger Receptor Expression in HumanMyeloid Lineage

To specifically determine which cells in the liver respond toPEG-rHuIL-10, in vitro experiments were conducted wherein humanmonocytes (FIG. 5A), human macrophages (FIG. 5B), human Kupffer cells(FIG. 5C), and human hepatocytes (FIG. 5D) were exposed to PEG-rHuIL-10.Referring to FIGS. 5A-5D, only the Kupffer cells and peripheralmonocytes showed an upregulation of scavenger receptor expression inresponse to PEG-rHuIL-10.

These data suggest that PEG-rHuIL-10 upregulates scavenger receptorexpression on myeloid lineage cells, but not hepatocytes. Of note,scavenger receptor regulation appeared to be similar in mice and humans,suggesting a conservation of IL-10's biology with regard to effects onthe myeloid compartment.

EXAMPLE 6 PEG-rHuIL-10 Increases Monocyte Uptake of Modified LDL and LDLUptake by Kupffer Cells

In order to determine if the increased expression of scavenger receptorscorrelates with enhanced uptake of lipoproteins, peripheral humanmonocytes (FIGS. 6A, 6E and 6F), human macrophages (FIG. 6B), humanKupffer cells (FIG. 6C), and human hepatocytes (FIG. 6D) were isolatedand exposed to PEG-rHuIL-10.

Referring to FIGS. 6A-6D, the Y-axis is the quantitation of the amountof labeled cholesterol that has been taken up relative to a standardcurve dilution of the same labeled cholesterol. The parameters on theX-axis can be briefly summarized as follows: cells were exposed toPEG-IL-10 for ˜24 hours, then washed and exposed for 3-5 hours to i)nothing with no labeled cholesterol to act as the background control,ii) nothing with the labeled cholesterol to act as the backgroundcontrol, or iii) PEG-IL-10 with the labeled cholesterol. Cells werewashed, lysed, and then the total internalized labeled cholesterol wasquantified as indicated above. The data in FIGS. 6E and 6F was generatedin a similar manner.

As indicated in FIG. 6A, PEG-rHuIL-10 increased the uptake of acetylatedLDL (Ac-LDL; *, p<0.05) and oxidized LDL (Ox-LDL; (***, p<0.001), butnot unmodified LDL (LDL), by freshly isolated peripheral bloodmonocytes. FIGS. 6E and 6F exhibit the effect of PEG-rHuIL-10 andPEG-rHuIL-10 plus mannose on acetylated LDL (FIG. 6E) and oxidized LDL(FIG. 6F) in human monocytes. Mannose was used to block the mannosereceptor. Blockade of the mannose receptor inhibited the uptake ofacetylated LDL, although not the uptake of oxidized LDL (FIGS. 6E and6F, respectively). These data suggest that PEG-rHuIL-10 enhances thescavenging of LDL proteins via the upregulation of both canonical andnon-canonical scavenger receptors.

In contrast, no differences were detected in any form of LDL uptake byMacrophage colony-stimulating factor (M-CSF)-differentiated macrophagesin response to PEG-rHuIL-10 (FIG. 6B). Referring to FIG. 6C,PEG-rHuIL-10 increased Kupffer cell uptake of LDL (*, p<0.05), but notAc-LDL or Ox-LDL. These data support the involvement of Kupffer cells inthe regulation of plasma LDL cholesterol. The presence of alterations tohepatocyte uptake of unmodified, oxidized and acetylated LDL was alsoassessed. In response to PEG-rHuIL-10, no alterations to hepatocyteuptake of unmodified, oxidized and acetylated LDL were observed (FIG.6D). While myeloid lineage cells are generally phagocytotic, these datashow that the nature of cholesterol uptake is different betweenmonocytes, macrophages, Kupffer cells, and hepatocytes in response toPEG-rHuIL-10.

When neutralizing antibodies to MARCO and CD36, as potential inhibitorsof PEG-rHuIL-10's effect on cholesterol uptake, were evaluated, nodifferences in uptake were observed (data not shown). In contrast,dextran inhibited PEG-rHuIL-10's increase in Ac-LDL but not Ox-LDLuptake by monocytes (data not shown). These data suggest thatPEG-rHuIL-10's control of lipoprotein uptake occurs through both knownand possibly unknown scavenger receptor regulation. Moreover, consistentwith efflux gene regulation in vivo, exposure of Kupffer cells to IL-10caused a moderate increase in efflux of cholesterol onto HDL particles(data not shown).

In their totality, these data indicate that while PEG-rHuIL-10 canenhance the uptake of Ac-LDL and Ox-LDL by peripheral monocytes, theKupffer cell population within the liver is the target cell populationfor PEG-rHuIL-10-mediated control of peripheral cholesterol.Furthermore, these data implicate the myeloid lineage in the control ofperipheral cholesterol, suggesting that monocytes, macrophages, andKupffer cells represent an untapped cellular reservoir within the liverthat actively removes plasma cholesterol. Finally, these data indicatethat the modulation of scavenger receptors on Kupffer cells withPEG-rHuIL-10, as well as the general modulation of myeloid lineage withother therapeutic compounds, may represent an alternative means forreducing plasma cholesterol.

EXAMPLE 7 PEG-rMuIL-10 is Additive with Ezetimibe in CholesterolLowering

In order to assess whether the PEG-rMuIL-10-mediated effects observed inthe previous examples were consistent with the utilization of a celltype not currently targeted by standard of care therapeutics,PEG-rMuIL-10 was combined with an orally administered statin orezetimibe, an oral therapeutic agent that blocks cholesterol absorption.

As illustrated in FIG. 7A, when administered to LDLR −/− mice on highfat diet, PEG-rMuIL-10 (1.0 mg/kg SC daily) and Ezetimibe (Ez) (10 mg/kgdaily via oral gavage) individually lowered plasma cholesterol by 58%and 55%, respectively, and by up to 86% when combined. The plasmacholesterol concentration observed with administration of PEG-rMuIL-10and Ezetimibe was similar to that of normal plasma cholesterol levelsobserved in LDLR −/− mice.

Data associated with plasma LDL-C and plasma HDL-C were generated usingan experimental approach comparable to that which gave rise to the datain FIG. 7A. As indicated in FIGS. 7A and 7B, combination of PEG-rMuIL-10and Ezetimibe resulted in reduction of LDL-C and HDL-C, respectively.Although combination of PEG-rMuIL-10 and statin (simvastatin) alsoreduced LDL-C and HDL-C (data not shown), oral administration of statinis toxic in mice, and therefore the maximal level of plasma loweringachieved with statin was limited. The combination of PEG-rMuIL-10 withboth Ezetimibe and statin suggests that the mechanism of action of eachagent is non-redundant.

EXAMPLE 8 PEG-rMuIL-10 Decreases the Accumulation of Lipids, Cholesteroland Triglycerides

An evaluation was conducted to determine the scavenger effect on plasmacholesterol of PEG-rMuIL-10-mediated activation of Kupffer cells.

As determined by assessment of hepatic lipid concentrations by Oil Red Ostaining (data not shown), treatment with PEG-rMuIL-10 decreased hepaticlipid droplets in LDLR−/− mice fed high fat chow (FIG. 8A). In addition,hepatic tissue concentrations of cholesterol and triglycerides werequantified. As illustrated in FIGS. 8B-8E, hepatic cholesterolconcentrations only trended lower in wt and LDLR−/− mice fed the highfat diet; mice receiving a normal diet did not exhibit an appreciableeffect. FIGS. 8E-8I indicated that hepatic triglyceride concentrationswere lower in the wt mice fed the high fat diet, and the LDLR−/− micefed both the both normal diet and high fat diet. In their totality,these data indicate that both i) the engagement of the scavenging systemwhereby plasma lipoproteins are removed by Kupffer cells, and ii) themoderately enhanced efflux onto HDL lipoproteins (data not shown), aresufficient to prevent inappropriate triglyceride and cholesterolaccumulation within the Kupffer cells and hepatocytes.

EXAMPLE 9 PEG-rMuIL-10 Treatment Results in Hepatocyte Proliferation

Administration of PEG-rMuIL-10 resulted in increased numbers ofperi-portal cells in LDLR−/− mice (data not shown), reflecting anapparent decrease in steatotic cells consistent with the decrease inliver triglycerides observed in the preceding example. As depicted inFIG. 9A, an increase in liver weight was observed that was diet- (normaldiet vs. high fat diet) and strain-independent (wt vs. LDLR−/−), butdose-related (data not shown). Ki67 gene expression was quantified inorder to determine whether that observation was reflective ofproliferation in the liver. As illustrated in FIGS. 9B-E, geneexpression was increased more than 2-fold in wt and LDLR−/− mice fed anormal diet and in wt and LDLR−/− mice fed a high fat diet when animalswere treated with 0.2 mg/kg PEG-rMuIL-10 (but not 0.02 mg/kgPEG-rMuIL-10).

In order to determine which cells were dividing, cells were stained forproliferating cell nuclear antigen (PCNA) by IHC, and an increase inPCNA-positive cells was observed in both wt mice fed normal chow andLDLR−/− mice fed high fat chow (data not shown). As indicated in FIGS.9F-9I PEG-rMuIL-10 dosing led to increases in PCNA-positive cells in allmice backgrounds. PCNA-positive cells appeared to be both hepatocytesand cells entering into the liver via the portal duct.

EXAMPLE 10 PEG-rMuIL-10 Exhibits an Anti-fibrotic Effect

Triglyceride accumulation in the liver is considered causative of fattyliver disease, and long-term accumulation has been linked to thedevelopment of NASH (see, e.g., Liu, Q., et al., Lipids Health Dis,2010. 9:42). Fatty liver disease is believed to progress to cirrhosis,which is associated with the deposition of collagen by activated hepaticstellate cells. IL-10 inhibits secretion of collagen from these cells.Therefore, an assessment was conducted to determine whether treatment ofnormal and hyperlipidemic wild type and LDLR−/− mice with PEG-rMuIL-10results in changes in hepatic collagen deposition.

Comparison of representative liver peri-portal regions from wt andLDLR−/− mice fed normal and high fat chow indicted that LDLR−/− mice feda high fat diet had the greatest changes to peri-portal collagendeposition (data not shown). These data suggest that treatment withPEG-rMuIL-10 may inhibit the deposition of peri-portal collagen,consistent with IL-10's reported anti-fibrotic function. Furtherassessment indicated that peri-portal collagen deposition in response tothe inflammatory stimulus derived from high plasma cholesterol can berestored to normal by PEG-rMuIL-10 dosing. Consistent with these data,PEG-rMuIL-10 also decreased liver triglycerides in these animals (datanot shown).

Taken together, these data suggest that PEG-rMuIL-10 may restore liverhealth by inducing hepatocyte proliferation, decreasing hepatictriglyceride concentrations, and remodeling inappropriately depositedcollagen. Accumulation of hepatic triglyceride and subsequent collagendeposition are hallmarks of NAFLD that, if left unchecked, develop intoNASH. Because treatment with PEG-rMuIL-10 reduces these causativefactors, PEG-IL-10 represents a potential therapeutic modality for thetreatment of NAFLD and NASH.

EXAMPLE 11 Effect of PEG-rMuIL-10 on Liver Function

A possible link between exposure of mice to PEG-rMuIL-10 andhepatotoxicity was explored. As set forth in Table 1, an assessment ofchanges to liver function serum markers indicated that PEG-rMuIL-10 didnot dramatically change liver serum function markers relative to normalmurine controls.

TABLE 1 Control 0.2 mg/kg Normal Liver Function Serum Median MedianMouse Marker Range Range Range ALT (IU/L) 20-74 34.5-122  17-77 AST(IU/L)  73-162  68-136  54-298 Albumin (g/dL)  2-2.9 2.4-3  2.5-3  TotalProtein (g/dL) 4.4-5.5 4.6-5.1 3.5-7.2 Alkaline Phosphatase (IU/L)89.5-121  26.5-49  3.5-96  Glucose (g/dL) 202-278 240-281  62-175 TotalBilirubin (mg/dL)   0-0.35 0.1-0.3  0-0.9 Phosphorus (mg/dL) 4.6-6.85.25-7.4  5.7-9.2 Blood Urea Nitrogen (mg/dL) 17.5-28  16-25  8-33Creatine Phosphokinase (IU/L) 109-417 172-329 105-649

Thus, while exposures of hypercholesterolemic mice to PEG-rMuIL-10resulted in certain changes in liver biology, those changes did notinduce toxic primary or secondary effects. These data further supportthe potential use of PEG-rHuIL-10 in controlling high plasma cholesteroland the concomitant beneficial effects on NAFLD and NASH.

Particular embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Upon reading the foregoing, description, variations of the disclosedembodiments may become apparent to individuals working in the art, andit is expected that those skilled artisans may employ such variations asappropriate. Accordingly, it is intended that the invention be practicedotherwise than as specifically described herein, and that the inventionincludes all modifications and equivalents of the subject matter recitedin the claims appended hereto as permitted by applicable law. Moreover,any combination of the above-described elements in all possiblevariations thereof is encompassed by the invention unless otherwiseindicated herein or otherwise clearly contradicted by context.

All publications, patent applications, accession numbers, and otherreferences cited in this specification are herein incorporated byreference as if each individual publication or patent application werespecifically and individually indicated to be incorporated by reference.

What is claimed is:
 1. A method of treating non-alcoholicsteatohepatitis (NASH) or non-alcoholic fatty liver disease (NAFLD) in asubject, the method comprising: administering parenterally to a subjecthaving NASH or NAFLD a therapeutically effective amount of a compositioncomprising a PEGylated interleukin-10 (PEG-IL-10) agent, wherein theamount is sufficient to maintain an IL-10 serum trough concentrationfrom 1.0 pg/mL to 10.0 ng/mL, over a period of time of at least 24hours.
 2. The method of claim 1, wherein the PEG-IL-10 agent comprisesmature human IL-10.
 3. The method of claim 1, wherein the PEG-IL-10agent comprises a variant of mature human IL-10, and wherein the variantexhibits activity comparable to the activity of mature human IL-10. 4.The method of claim 1, wherein said administering is effective todecrease cholesterol in the subject.
 5. The method of claim 1, whereinsaid administering is effective to decrease triglycerides in thesubject.
 6. The method of claim 1, wherein said administering iseffective to decrease peri-portal collagen deposition in the subject. 7.The method of claim 1, wherein said administering is effective toincrease hepatocyte proliferation in the subject.
 8. The method of claim1, wherein said administering is subcutaneous.
 9. The method of claim 1,wherein the method comprises administering at least one additionalprophylactic or therapeutic agent.
 10. The method of claim 1, whereinthe PEG-IL-10 agent comprises at least one PEG molecule covalentlyattached to at least one amino acid residue of at least one subunit ofIL-10.
 11. The method of claim 1, wherein the PEG-IL-10 agent comprisesa mixture of mono-pegylated and di-pegylated IL-10.
 12. The method ofclaim 1, wherein the PEG component of the PEG-IL-10 agent has amolecular mass from about 5 kDa to about 20 kDa.
 13. The method of claim1, wherein the PEG component of the PEG-IL-10 agent has a molecular massgreater than about 20 kDa.
 14. The method of claim 1, wherein the PEGcomponent of the PEG-IL-10 agent has a molecular mass of at least about30 kD.
 15. The method of claim 1, wherein the subject is a human.
 16. Amethod of treating hypercholesterolemia or ahypercholesterolemia-associated disease, disorder or condition in asubject having non-alcoholic steatohepatitis (NASH) or non-alcoholicfatty liver disease (NAFLD), the method comprising: administeringparenterally to the subject a composition comprising PEGylatedinterleukin-10 (PEG-IL-10) agent and a composition comprising ezetimibe,wherein said administering is effective to reduce cholesterol in thesubject and wherein the amount is sufficient to maintain an IL-10 serumtrough concentration from 1.0 pg/mL to 10.0 ng/mL, over a period of timeof at least 24 hours.
 17. The method of claim 16, wherein thecomposition comprising PEG-IL-10 and the composition comprising aezetimibe are administered sequentially.
 18. The method of claim 16,wherein the PEG-IL-10 agent comprises mature human IL-10.
 19. The methodof claim 16, wherein the PEG-IL-10 agent comprises a variant of maturehuman IL-10, and wherein the variant exhibits activity comparable to theactivity of mature human IL-10.
 20. The method of claim 16, wherein saidadministering of the composition comprising the PEG-IL-10 agent issubcutaneous.
 21. The method of claim 16, wherein the PEG-IL-10 agentcomprises at least one PEG molecule covalently attached to at least oneamino acid residue of at least one subunit of IL-10.
 22. The method ofclaim 16, wherein the PEG-IL-10 agent comprises a mixture ofmono-pegylated and di-pegylated IL-10.
 23. The method of claim 16,wherein the PEG component of the PEG-IL-10 agent has a molecular massfrom about 5 kDa to about 20 kDa.
 24. The method of claim 16, whereinthe PEG component of the PEG-IL-10 agent has a molecular mass greaterthan about 20 kDa.
 25. The method of claim 16, wherein the PEG componentof the PEG-IL-10 agent has a molecular mass of at least about 30 kD. 26.The method of claim 16, wherein the subject is a human.